The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016

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Au cours des dernières années, le développement des connaissances au niveau de l’étiologie de la maladie ainsi que l’arrivée de nouveaux médicaments et de lignes directrices guidant la pratique clinique sont susceptibles d’avoir entraîné une meilleure gestion de la polyarthrite rhumatoïde (PAR) et de l’ostéoporose, une comorbidité fréquente chez ces patients. Dans cette thèse, trois questions de recherche sont étudiées à l’aide des banques de données administratives québécoises (RAMQ, MED-ÉCHO). Une première étude documente l’utilisation des médicaments pour la PAR au Québec. À ce jour, il s’agit de la seule étude canadienne à rapporter les tendances d’utilisation des DMARD (disease-modifying antirheumatic drug) biologiques depuis leur introduction dans la pratique clinique. Au cours de la période à l’étude (2002-2008), l’utilisation de DMARD (synthétiques et biologiques) a augmenté légèrement dans la population atteinte de PAR (1,9%, 95% CI : 1,1 - 2,8). Cependant, malgré la présence de recommandations cliniques soulignant l’importance de commencer un traitement rapidement, et la couverture de ces traitements par le régime général d’assurance médicaments, les résultats démontrent une initiation sous-optimale des DMARD chez les patients nouvellement diagnostiqués (probabilité d’initiation à 12 mois : 38,5%). L’initiation de DMARD était beaucoup plus fréquente lorsqu’un rhumatologue était impliqué dans la provision des soins (OR : 4,31, 95% CI : 3,73 - 4,97). Concernant les DMARD biologiques, le facteur le plus fortement associé avec leur initiation était l’année calendrier. Chez les sujets diagnostiqués en 2002, 1,2 sur 1 000 ont initié un DMARD biologique moins d’un an après leur diagnostic. Pour ceux qui ont été diagnostiqués en 2007, le taux était de 13 sur 1 000. Les résultats démontrent que si la gestion pharmacologique de la PAR s’est améliorée au cours de la période à l’étude, elle demeure tout de même sous-optimale. Assurer un meilleur accès aux rhumatologues pourrait, semble-t-il, être une stratégie efficace pour améliorer la qualité des soins chez les patients atteints de PAR. Dans une deuxième étude, l’association entre l’utilisation des DMARD biologiques et le risque de fractures ostéoporotiques non vertébrales chez des patients PAR âgés de 50 ans et plus a été rapportée. Puisque l’inflammation chronique résultant de la PAR interfère avec le remodelage osseux et que les DMARD biologiques, en plus de leur effet anti-inflammatoire et immunosuppresseur, sont des modulateurs de l’activité cellulaire des ostéoclastes et des ostéoblastes pouvant possiblement mener à la prévention des pertes de densité minérale osseuse (DMO), il était attendu que leur utilisation réduirait le risque de fracture. Une étude de cas-témoin intra-cohorte a été conduite. Bien qu’aucune réduction du risque de fracture suivant l’utilisation de DMARD biologiques n’ait pu être démontrée (OR : 1,03, 95% CI : 0,42 - 2,53), l’étude établit le taux d’incidence de fractures ostéoporotiques non vertébrales dans une population canadienne atteinte de PAR (11/1 000 personnes - années) et souligne le rôle d’importants facteurs de risque. La prévalence élevée de l’ostéoporose dans la population atteinte de PAR justifie que l’on accorde plus d’attention à la prévention des fractures. Finalement, une troisième étude explore l’impact de la dissémination massive, en 2002, des lignes directrices du traitement de l’ostéoporose au Canada sur la gestion pharmacologique de l’ostéoporose et sur les taux d’incidence de fractures ostéoporotiques non vertébrales chez une population de patients PAR âgés de 50 ans et plus entre 1998 et 2008. Étant donné la disponibilité des traitements efficaces pour l’ostéoporose depuis le milieu des années 1990 et l’évolution des lignes directrices de traitement, une réduction du taux de fractures était attendue. Quelques études canadiennes ont démontré une réduction des fractures suivant une utilisation étendue des médicaments contre l’ostéoporose et de l’ostéodensitométrie dans une population générale, mais aucune ne s’est attardée plus particulièrement sur une population adulte atteinte de PAR. Dans cette étude observationnelle utilisant une approche de série chronologique, aucune réduction du taux de fracture après 2002 (période suivant la dissémination des lignes directrices) n’a pu être démontrée. Cependant, l’utilisation des médicaments pour l’ostéoporose, le passage d’ostéodensitométrie, ainsi que la provision de soins pour l’ostéoporose en post-fracture ont augmenté. Cette étude démontre que malgré des années de disponibilité de traitements efficaces et d’investissement dans le développement et la promotion de lignes directrices de traitement, l’effet bénéfique au niveau de la réduction des fractures ne s’est toujours pas concrétisé dans la population atteinte de PAR, au cours de la période à l’étude. Ces travaux sont les premiers à examiner, à l’aide d’une banque de données administratives, des sujets atteints de PAR sur une période s’étalant sur 11 ans, permettant non seulement l’étude des changements de pratique clinique suivant l’apparition de nouveaux traitements ou bien de nouvelles lignes directrices, mais également de leur impact sur la santé. De plus, via l’étude des déterminants de traitement, les résultats offrent des pistes de solution afin de combler l’écart entre la pratique observée et les recommandations cliniques. Enfin, les résultats de ces études bonifient la littérature concernant la qualité des soins pharmacologiques chez les patients PAR et de la prévention des fractures. / Over the past two decades, progresses in the understanding of disease etiology, the arrival of new drugs, and the development of clinical practice guidelines may have led to a better pharmacological management of rheumatoid arthritis (RA) and osteoporosis, a common comorbidity. In this thesis, three research questions were investigated using well characterized administrative databases (RAMQ and MED-ECHO). A first study documented RA drug use in the province of Quebec. To this date, this is the only Canadian study to report on patterns of biologic DMARD use since their introduction in clinical practice. Over our study time horizon (2002-2008), the use of any DMARDs (synthetic and biologic) slightly increased in the overall RA population (1.9%, 95% CI: 1.1-2.8). However, despite clinical practice guidelines stressing the importance of early treatment, and the reimbursement of treatments by the Quebec drug plan, the results demonstrated suboptimal DMARD initiation in newly diagnosed RA (probability at 12 months: 38.5%), though DMARD initiation increased when rheumatologists were overseeing care (OR: 4.31, 95%CI: 3.73-4.97). For biologic DMARDs, the strongest predictor of initiation was the calendar year of study entry. Of subjects newly diagnosed in 2002, 1.2 in 1000 had a biologic initiated within one year, while for those newly diagnosed in 2007, it was 13.0 in 1000. The results showed that the pharmacological management of RA is improving over time, but remains below expectations. Ensuring better access to rheumatologists should be an area of focus in order to enhance the quality of RA care. A second study reported on biologic DMARD use and the risk of non-vertebral osteoporotic fractures in RA patients aged ≥50 years. Because chronic inflammation in RA interferes with bone remodeling and biologic DMARDs, in addition to their anti-inflammatory and immunosuppressive effects, are modulators of the cellular activity of osteoblasts and osteoclasts possibly leading to the preservation of bone mineral density (BMD), it was believed that their use may reduce the risk of fractures. A nested-case control study was conducted. Although a reduction in the risk of fractures subsequent to biologic DMARD use could not be demonstrated (OR: 1.03, 95% CI: 0.42-2.53), the study established the incidence rate of non-vertebral osteoporotic fractures in a Canadian RA population (11/1000 person-years) and highlighted some important risk factors. The high prevalence of osteoporosis in the RA population justifies that more attention be paid to preventing fractures. Finally, a third study investigated the impact of the 2002 Canadian osteoporosis guidelines on the pharmacological management of osteoporosis and on the rates of non-vertebral osteoporotic fractures in a RA population aged ≥50 years between 1998 and 2008. With the availability of effective osteoporosis treatments since the mid 90s, and the evolving clinical practice guidelines, a reduction in the rate of fractures was expected. Some Canadian studies have shown reductions in the rate of fractures following broader use of osteoporosis drugs and BMD testing in a general adult population, but none have specifically investigated the impact in RA. In this observational study using a time series approach, no reduction in the rate of fractures after 2002 (post guidelines dissemination) could be demonstrated. However, the use of osteoporosis drugs, BMD testing, and provision of post fracture osteoporosis care improved. This study demonstrated that years of availability of effective preventive measures and investments in the development and promotion of clinical practice guidelines have not yet translated into further reduction in the rate of fractures in our RA population over our study time horizon. This body of work is the first to examine, using healthcare administrative data, subjects with RA over a period of 11 years, allowing not only to study the changes in clinical practice following the introduction of new treatments and guidelines, but also to capture the impact on health. In addition, by studying predictors of treatment, the results provide good insights in terms of solutions to fill the gap between the observed clinical practice and guideline recommendations. Finally, the results of these studies substantiate the literature regarding the quality of RA care and the prevention of fractures.

Distributed systems are everywhere. From large datacenters to mobile devices, an ever richer assortment of applications and services relies on distributed systems, infrastructure, and protocols. Despite their ubiquity, testing and debugging distributed systems remains notoriously hard. Moreover, aside from inherent design challenges posed by partial failure, concurrency, or asynchrony, there remain significant challenges in the implementation of distributed systems. These programming challenges stem from the increasing complexity of the concurrent activities and reactive behaviors in a distributed system on the one hand, and the need to effectively leverage the parallelism offered by modern multi-core hardware, on the other hand. This thesis contributes Kompics, a programming model designed to alleviate some of these challenges. Kompics is a component model and programming framework for building distributed systems by composing message-passing concurrent components. Systems built with Kompics leverage multi-core machines out of the box, and they can be dynamically reconfigured to support hot software upgrades. A simulation framework enables deterministic execution replay for debugging, testing, and reproducible behavior evaluation for large-scale Kompics distributed systems. The same system code is used for both simulation and production deployment, greatly simplifying the system development, testing, and debugging cycle. We highlight the architectural patterns and abstractions facilitated by Kompics through a case study of a non-trivial distributed key-value storage system. CATS is a scalable, fault-tolerant, elastic, and self-managing key-value store which trades off service availability for guarantees of atomic data consistency and tolerance to network partitions. We present the composition architecture for the numerous protocols employed by the CATS system, as well as our methodology for testing the correctness of key CATS algorithms using the Kompics simulation framework. Results from a comprehensive performance evaluation attest that CATS achieves its claimed properties and delivers a level of performance competitive with similar systems which provide only weaker consistency guarantees. More importantly, this testifies that Kompics admits efficient system implementations. Its use as a teaching framework as well as its use for rapid prototyping, development, and evaluation of a myriad of scalable distributed systems, both within and outside our research group, confirm the practicality of Kompics. / Kompics / CATS / REST

Em ecologia, o comportamento dos animais é freqüentemente estudado para entender melhor suas preferências por diferentes tipos de alimento e habitat. O presente trabalho esta relacionado a este tópico, dividindo-se em três capítulos. O primeiro capitulo refere-se à estimação da função da probabilidade da seleção de recurso (RSPF) comparado com um modelo de escolha discreta (DCM) com uma escolha, usando as estatísticas qui-quadrado para obter as estimativas. As melhores estimativas foram obtidas pelo método DCM com uma escolha. No entanto, os animais não fazem a sua seleção baseados apenas em uma escolha. Com RSPF, as estimativas de máxima verossimilhança, usadas pela regressão logística ainda não atingiram os objetivos, já que os animais têm mais de uma escolha. R e o software Minitab e a linguagem de programação Fortran foram usados para obter os resultados deste capítulo. No segundo capítulo discutimos mais a verossimilhança do primeiro capítulo. Uma nova verossimilhança para a RSPF é apresentada, a qual considera as unidades usadas e não usadas, e métodos de bootstrapping paramétrico e não paramétrico são usados para estudar o viés e a variância dos estimadores dos parâmetros, usando o programa FORTRAN para obter os resultados. No terceiro capítulo, a nova verossimilhança apresentada no capítulo 2 é usada com um modelo de escolha discreta, para resolver parte do problema apresentado no primeiro capítulo. A estrutura de encaixe é proposta para modelar a seleção de habitat de 28 corujas manchadas (Strix occidentalis), assim como a uma generalização do modelo logit encaixado, usando a maximização da utilidade aleatória e a RSPF aleatória. Métodos de otimização numérica, e o sistema computacional SAS, são usados para estimar os parâmetros de estrutura de encaixe. / In ecology, the behavior of animals is often studied to better understand their preferences for different types of habitat and food. The present work is concerned with this topic. It is divided into three chapters. The first concerns the estimation of a resource selection probability function (RSPF) compared with a discrete choice model (DCM) using chi-squared to obtain estimates. The best estimates were obtained by the DCM method. Nevertheless, animals were not selected based on choice alone. With RSPF, the maximum likelihood estimates used with the logistic regression still did not reach the objectives, since the animals have more than one choice. R and Minitab software and the FORTRAN programming language were used for the computations in this chapter. The second chapter discusses further the likelihood presented in the first chapter. A new likelihood for a RSPF is presented, which takes into account the units used and not used, and parametric and non-parametric bootstrapping are employed to study the bias and variance of parameter estimators, using a FORTRAN program for the calculations. In the third chapter, the new likelihood presented in chapter 2, with a discrete choice model is used to resolve a part of the problem presented in the first chapter. A nested structure is proposed for modelling selection by 28 spotted owls (Strix occidentalis) as well as a generalized nested logit model using random utility maximization and a random RSPF. Numerical optimization methods and the SAS system were employed to estimate the nested structural parameters.

分析具有巢狀誤差結構的迴歸模式時，惹忽略隨機誤差項之間的相關性，而採用最小平方(OLS)估計量所導出的標準 F 統計量（以 F^S表之）進行檢定，會導致過大的型 I 錯誤機率；若將隨機誤差項之間的相關性納入考量，而採用廣義最小平方(GLS)估計量所導出的 F 統計量 （以 F^GLS表之），則計算上會較為繁雜。因此我們藉由轉換方式，將模式轉換成隨機誤差項之間彼此獨立的新模式後，再以 F^S 進行檢定，其結果與直接以 F^GLS 檢定相同，且可使計算較為方便。由於模式轉換所需的轉換矩陣為母體變異數的函數，因此當母體變異數未知時，我們以 Henderson 的常數配適 （fitting-of-constants）方法來估計之。藉由模擬結果得知，若各段的觀察個數相等，則不論巢狀誤差結構為二段式(two-stage)或三段式(three-stage)，廣義最小平方估計量(GLS)均較最小平方估計量(OLS)表現穩定，且 F^GLS 在檢定力及實際顯著水準方面的表現也都比 F^S 好。 / When analyzing the regression model with nested-error structure, if the correlations between errors are ignored, and conduting the model adequacy test by the standard F statistic (F^S) led from the ordinary leastsquares estimator (OLSE) , then the type I error rate will be inflated. However, if the corrlated structure is considered and the model is tested by F^GLS led from the general least-squares estimator (GLSE) , the calculation will be more complicate. The model can be transformed to a new model with independent random errors and then, tested by F^S . The result is the same as the one by F^GLS , also it is more convenient for calculation. Since the transformation matrix is a function of variance components, we estimate variance components by Henderson's fitting-of-constants when they are unknown. Through simulation, it is concluded that if the observations in each stage of nested-error structure are the same, the GLSE is more stable than the OLSE in both two-stage and tree-stage structures. Also, the power and the sizes of F^GLS will perform better than those of F^S .

在當前的治理環境下，公共任務比以往更需要整合政府及各部門組織的行動方能克盡其功。因此，多數的公共管理者應會同意，行政組際協調已成為「治理時代」重要且迫切的議題之一，公共管理者需要擁有全新的能力，從解決民眾問題的角度來回應民眾需求。本文將行政組際協調定義為「藉由兩個或兩個以上行政組織的一致行動，使特定政策或計畫的執行，能達成最少的冗餘、不一致與空隙的執行結果」。在此定義下，本文討論的行政組際協調涉及三個層面：第一個層面為跨行政組織如何產生一致行動的問題；第二個層面為行政組織間的互動關係；第三個層面為跨行政組織執行成果的問題。 本文建構的「行政組際協調嵌套賽局模型」假定官僚制度中的專業分工與獲利轉換機制的制度設計，是造成行政組織分工但不合作的主因。在此前提下，筆者引入「效用損失」的概念，做為發展行政組織行為效用函數的基礎。在行政自主性「效用損失」的概念下，筆者僅保留與行政組織政策或計畫執行最相關的自變項，分別是相依關係、溝通、管轄領域、民意監督、外部課責與內部課責等六種，來解釋行政組織的合作行為以及協調的結果等兩種依變項。由於本文將制度視為對參賽者的限制與機會，在制度陳述概念的輔助下，筆者得以清楚地設定行政組際協調的賽局情境，並將行政責任的思考轉化為外部課責與內部課責等兩種課責參數型態。在此課責制度框架下，筆者建立行政組際協調的空間結構，透過行政組織自主性效用之簡單損失函數以及制度空間模型的運用，成功建立起一個階層管理者、兩個行政組織的行政組際協調嵌套賽局模型。這個模型依據外部課責是否一致，以及內部課責是否存有共識等兩個面向，將行政組際協調賽局情境區分為四種類型，並在分別推演參賽者的行為變化後，提出十項理論命題。為詮釋這些命題在現實環境中的意義，筆者在臺北市政府研考會的同意下，引用該會於2010年10月辦理之1999跨機關陳情案件問卷調查資料，進行次級資料分析。 綜合而言，本文建構的「行政組際協調嵌套賽局模型」，是建立在一個嚴格的假定條件之上的，因此其理論的解釋力與預測能力都僅能限縮在一定的範圍內，特別是一階層管理者、兩行政組織的三人完全訊息賽局。換言之，超出這個範圍之外的行政組際協調現象，就不適合使用本模型進行解釋。本文雖然只使用極精簡的相關研究變項，卻也足以展現一個理論模型應具備的解釋與預測能力。當然，本文的研究僅是一個開端，不論在模型的廣博性以及適用性都還有極大的待改善空間。筆者也鼓勵後繼的學者，能持續地擴展與修改本文提出之理論模型，讓行政組際協調研究領域能朝向更正面的發展。 / Under the present governance environment, the government would need more efforts to coordinate different organizations’ actions than before to make sure the public services would be provided successfully. Thus, most public managers would not only agree that the interorganizational coordination has become one of the important and urgent issues in the governance era, but also they need to learn new abilities to response the citizens’ needs. The author defined the concept of interorganizational coordination as “The end-state of a public policy or program which is implemented by two or more organizations in a consistent way is characterized by minimal redundancy, incoherence and lacunae.” Under this definition, the author discussed three different questions of interorganization coordination in public administration. The first question is How can we formulate a set of consistent actions for implementing a public policy or program? The second question is “How can we explain the interactive relationship between the organizations in public administration?” The third question is “What kind of results would be produced by multi-organizational implementation?” The nested game model of this dissertation has been assumed that the specification and unique side payment system of bureaucracy are the fundamental institution of interorganizational coordination. Under this assumption, the author introduced the concept of simple loss function and structure-induced equilibrium to create an utility function of public organizations and a spatial model for deducing propositions of interorganizational coordination in public administration. In order to verify the propositions of the nested game model of this dissertation, the author did a case study which was including 52 appealed cases of 1999 Citizen Hotline of Taipei City Government and tested the hypothesis derived from the propositions. Finally, the author concluded that there are six independent variables, including interdependency, communication, territory, supervision, outside accountability and inside accountability which can be used to explain two dependent variables, including cooperative behaviors and the result of interorganizational coordination. The author admitted that the interorganizational coordination is a contingent process and should be carefully defined its game rules before discussing what happened in this process. This dissertation has provided a simplicity model for explaining interorganizational coordination with one hierarchical organization and two horizontal organizations within four different situations. The author hoped that other researchers can modify this simple model to explain more complex situations of interorganizational coordination. Thus, this field could be continually developed in a positive way.

Distributed systems are everywhere. From large datacenters to mobile devices, an ever richer assortment of applications and services relies on distributed systems, infrastructure, and protocols. Despite their ubiquity, testing and debugging distributed systems remains notoriously hard. Moreover, aside from inherent design challenges posed by partial failure, concurrency, or asynchrony, there remain significant challenges in the implementation of distributed systems. These programming challenges stem from the increasing complexity of the concurrent activities and reactive behaviors in a distributed system on the one hand, and the need to effectively leverage the parallelism offered by modern multi-core hardware, on the other hand. This thesis contributes Kompics, a programming model designed to alleviate some of these challenges. Kompics is a component model and programming framework for building distributed systems by composing message-passing concurrent components. Systems built with Kompics leverage multi-core machines out of the box, and they can be dynamically reconfigured to support hot software upgrades. A simulation framework enables deterministic execution replay for debugging, testing, and reproducible behavior evaluation for largescale Kompics distributed systems. The same system code is used for both simulation and production deployment, greatly simplifying the system development, testing, and debugging cycle. We highlight the architectural patterns and abstractions facilitated by Kompics through a case study of a non-trivial distributed key-value storage system. CATS is a scalable, fault-tolerant, elastic, and self-managing key-value store which trades off service availability for guarantees of atomic data consistency and tolerance to network partitions. We present the composition architecture for the numerous protocols employed by the CATS system, as well as our methodology for testing the correctness of key CATS algorithms using the Kompics simulation framework. Results from a comprehensive performance evaluation attest that CATS achieves its claimed properties and delivers a level of performance competitive with similar systems which provide only weaker consistency guarantees. More importantly, this testifies that Kompics admits efficient system implementations. Its use as a teaching framework as well as its use for rapid prototyping, development, and evaluation of a myriad of scalable distributed systems, both within and outside our research group, confirm the practicality of Kompics. / <p>QC 20130520</p>

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