困難梭狀桿菌是醫院內抗生素相關腹瀉的首要原因，也被稱為困難梭菌相關疾病，此外也是偽膜性結腸炎的主要病原體。在嚴重的案例中，困難梭菌感染(CDI)會造成毒性巨結腸症、腸穿孔甚至死亡。腸道上皮細胞是困難梭菌黏附與定殖過程中最先接觸到的組織，並且是對抗細菌感染的第一道物理防線。而除了被廣泛研究的細胞毒性機制以外，宿主細胞與困難梭菌之間的全基因組交互作用機制卻鮮少被探討。在過去20年，CDI的發病率與嚴重程度都有顯著的上升，而在低風險族群中的病例上升也促使我們尋找其他的致病機制與毒性因子。因此，我們的目標是觀察人類結腸上皮Caco-2細胞與困難梭菌細胞於感染早期(0~60 min)與感染後期(30~120 min)所進行的分子訊號耦合機制。
在這篇文章中，我們使用一種系統生物學方法來探究感染過程中的訊號耦合機制，此種系統生物學方法包括了大數據資料探勘、動態網路模型、全基因組資料鑒定方法、系統維度檢測以及主要網路投影(PNP)。我們依此建立全基因組基因與表觀遺傳網路(GEINs)並從其中抽取出宿主-病原體核心網路(HPCNs)來研究早期與晚期CDI之間分子訊號耦合機制的演進。基於我們的結果，我們建議細胞壁蛋白CD2787和CD0237可以做為潛在的藥物標靶，因為這兩者在細胞黏附以及細菌防衛機制中都扮演了重要的角色。此外，困難梭菌進行芽孢化的重要蛋白例如CD1214、CD2629和CD2643也可以作為藥物標靶，因為芽胞所導致的二次感染是CDI中的一個重要議題。最後，我們提出一種多分子藥物，包含了E64、CD0237-specific IgY以及REP3123來治療CDI，這些藥物能抑制上述的病原體基因來達到治療的目的。

Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, known as C. difficile-associated disease and the major etiologic agent of pseudomembranous colitis. In severe cases, C. difficile infection (CDI) can induce toxic megacolon, intestinal perforation and even death. The intestinal epithelial cells are the first tissue to be encountered for the adhesion and colonization of C. difficile, and play as the first physical defense barrier against infection. Despite the well characterized cytotoxicity, few studies investigate the genome-wide interplay between host cells and C. difficile. It has been shown a significant increase of the morbidity and severity of CDI in the past two decades, and the increasing cases in low risk groups also urge us to find other crucial pathogenic mechanisms and virulence factors. Thus, we aim to investigate cross-talk genetic and epigenetic molecular mechanisms between human colorectal epithelial Caco-2 cells and C. difficile during the early-stage (0~60 min) and the late-stage (30~120 min) of infection.
In this study, we introduced a systems biology approach to investigate the cross-talk mechanisms during the progression of infection via big data mining, dynamic network modeling, genome-wide data identification method, system order detection scheme and principal network projection method (PNP). We thus focus on how to construct genome-wide genetic-and-epigenetic interspecies networks (GEINs) and then extract host-pathogen core networks (HPCNs) to investigate the progression of underlying host/pathogen cross-talk genetic and epigenetic mechanisms from the early-stage to the late-stage of CDI. Based on our results, we suggest that the cell wall proteins CD2787 and CD0237, which both play an important role in cell adhesion and pathogen defense mechanisms, can be considered as potential drug targets. In addition, the crucial proteins employed by C. difficile for sporulation, including CD1214, CD2629 and CD2643, can also be considered as potential drug targets since the spore-mediated re-infection is an urgent issue to be solved. Finally, we proposed a potential multi-molecule drug containing E64, CD0237-specific IgY and REP3123 for the treatment of CDI due to their inhibitory abilities toward above targets.

誌謝 I
摘要 II
Abstract III
Contents IV
List of Tables VI
List of Figures VI
List of Supplemental materials VI
Introduction 1
Materials and Methods 4
2.1 Overview of the construction of GEINs and HPCNs in Caco-2 cells during the early-stage and late-stage of CDI 4
2.2 Big data mining and data preprocessing of host/pathogen gene/miRNA microarray data 4
2.3 Construction of candidate GEIN 5
2.4 Dynamic models of GEINs for Caco-2 cells and C. difficile during the infection 6
2.5 Parameter estimation of the dynamic models of candidate GEIN via system identification method 10
2.6 Pruning false-positives in candidate GEIN for real GEIN via system order detection scheme 17
Results 23
3.1 The identified GEINs at the early stage and late stage of Clostridium difficile infection 23
3.2 The host-pathogen core networks (HPCNs) during the infection of C. difficile 23
3.2.1 Construction of HPCNs to investigate the epigenetic activities in host core networks of CDI 23
3.2.2 Comparison of the pathogen core network with previously predicted core genes in C. difficile 25
3.2.3 Cross-talk mechanisms among host-pathogen interactions and their validations 26
3.3 A precise view of pathogenic effects and host responses at the early stage of C. difficile invasion 29
3.3.1 Pathogenic factors utilized by C. difficile and the resulting pathogenesis in Caco-2 cells…………………………………………………………………………………………………………………………….29
3.3.2 Caco-2 cells adopt autophagy, DNA damage response and the activation of PAK1 and GRB2 as remedial schemes in response to pathogen-induced damage 31
3.3.3 The offensive mechanisms of Caco-2 cells and the defense mechanisms of C. difficile at the early stage of CDI 32
3.4 The strong cellular-activities of Caco-2 cells and the infection results of host and pathogen at the late stage of infection 34
3.4.1 The emphasized ROS-production and stress-accumulation of host cells, and the failure of antioxidative defense mechanisms in C. difficile 34
3.4.2 The apoptosis process triggered by severe inflammation and accumulated cellular stresses of Caco-2 cells and the leaving of C. difficile via endospore-formation 37
3.5 Drug targets prediction and multi-molecule drug design for treating CDI 39
Discussion 41
Conclusion 44

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