Development in systems biology research has accelerated in recent years, and the reconstructions for molecular networks can provide a global view to enable in-depth investigation on numerous system ...properties in biology. However, we still lack a systematic approach to reconstruct the dynamic protein-protein association networks at different time stages from high-throughput data to further analyze the possible cross-talks among different signaling/regulatory pathways. In this study we integrated protein-protein interactions from different databases to construct the rough protein-protein association networks (PPANs) during TNFa-induced inflammation. Next, the gene expression profiles of TNFa-induced HUVEC and a stochastic dynamic model were used to rebuild the significant PPANs at different time stages, reflecting the development and progression of endothelium inflammatory responses. A new cross-talk ranking method was used to evaluate the potential core elements in the related signaling pathways of toll-like receptor 4 (TLR-4) as well as receptors for tumor necrosis factor (TNF-R) and interleukin-1 (IL-1R). The highly ranked cross-talks which are functionally relevant to the TNFa pathway were identified. A bow-tie structure was extracted from these cross-talk pathways, suggesting the robustness of network structure, the coordination of signal transduction and feedback control for efficient inflammatory responses to different stimuli. Further, several characteristics of signal transduction and feedback control were analyzed. A systematic approach based on a stochastic dynamic model is proposed to generate insight into the underlying defense mechanisms of inflammation via the construction of corresponding signaling networks upon specific stimuli. In addition, this systematic approach can be applied to other signaling networks under different conditions in different species. The algorithm and method proposed in this study could expedite prospective systems biology research when better experimental techniques for protein expression detection and microarray data with multiple sampling points become available in the future.
Signal transduction is the major mechanism through which cells transmit external stimuli to evoke intracellular biochemical responses. Diverse cellular stimuli create a wide variety of transcription ...factor activities through signal transduction pathways, resulting in different gene expression patterns. Understanding the relationship between external stimuli and the corresponding cellular responses, as well as the subsequent effects on downstream genes, is a major challenge in systems biology. Thus, a systematic approach is needed to integrate experimental data and theoretical hypotheses to identify the physiological consequences of environmental stimuli.
We proposed a systematic approach that combines forward and reverse engineering to link the signal transduction cascade with the gene responses. To demonstrate the feasibility of our strategy, we focused on linking the NF-kappaB signaling pathway with the inflammatory gene regulatory responses because NF-kappaB has long been recognized to play a crucial role in inflammation. We first utilized forward engineering (Hybrid Functional Petri Nets) to construct the NF-kappaB signaling pathway and reverse engineering (Network Components Analysis) to build a gene regulatory network (GRN). Then, we demonstrated that the corresponding IKK profiles can be identified in the GRN and are consistent with the experimental validation of the IKK kinase assay. We found that the time-lapse gene expression of several cytokines and chemokines (TNF-alpha, IL-1, IL-6, CXCL1, CXCL2 and CCL3) is concordant with the NF-kappaB activity profile, and these genes have stronger influence strength within the GRN. Such regulatory effects have highlighted the crucial roles of NF-kappaB signaling in the acute inflammatory response and enhance our understanding of the systemic inflammatory response syndrome.
We successfully identified and distinguished the corresponding signaling profiles among three microarray datasets with different stimuli strengths. In our model, the crucial genes of the NF-kappaB regulatory network were also identified to reflect the biological consequences of inflammation. With the experimental validation, our strategy is thus an effective solution to decipher cross-talk effects when attempting to integrate new kinetic parameters from other signal transduction pathways. The strategy also provides new insight for systems biology modeling to link any signal transduction pathways with the responses of downstream genes of interest.
博士
國立清華大學
生物資訊與結構生物研究所
101
The zebrafish model has become one of the best research systems for high throughput drug screening. The importance of zebrafish model in human disease research and for drug ...discovery and development is well-recognized in the past decade. To understand the pathogenesis of infectious and inflammatory diseases, many zebrafish disease models were established, including bacterial or viral infection diseases, autoimmune encephalomyelitis and colitis. Some of the models have been applied for drug screening to evaluate the therapeutic effect of novel compounds. Besides, the zebrafish model can be integrated with image-based assays, which can transform into an automated drug-evaluating platform. Such integration of disease model and screening platform can greatly advance our understanding of immunity-related pathogenesis and to improve the drug development.
By applying histological, gene expression and survival assays, we are the first group to establish a fungal infection model with zebrafish to investigate Candida albicans’ virulence, especially its hyphal formation during infection. We demonstrated that C. albicans could colonize and invade zebrafish at multiple anatomical sites and kill the fish in a dose-dependent manner. Moreover, using zebrafish embryos, we monitored C. albicans infection and visualized the interaction between pathogen and host myelomonocytic cells in vivo. Inside zebrafish, we observed the progression of the C. albicans yeast-to-hypha transition by tracking morphogenesis, and we monitored the corresponding genes expression of the pathogen. We then performed time-lapse microarray study to analyze the genes expression patterns in fungal pathogen and zebrafish host simultaneously for systems biology approach analysis, which the abovementioned is the first part of this thesis.
There are two parts in my thesis and the second part is to present how we modified a published protocol to induce inflammatory bowel diseases (IBD) in zebrafish larva by incubating fish in 2,4,6-trinitrobenzenesulfonic acid (TNBS) contained solution and apply the protocol for drug screening to select immunomodulatory molecules. By incorporating IBD scoring system and genes expression assays, we selected effective candidates from tryptophan metabolites in the activation of aryl hydrocarbon receptor (AhR) signaling to protect fish from IBD and we found the uremic toxic, indoxyl sulfate (IS), possess a physiological function to promote the expression of interleukin 22 to protect fish gut from IBD instead to damaging the physiological homeostasis. This finding proposed the IS can play a role to maintain the immunological balance, which is other than what had been reported as a toxic molecules.
In conclusion, our findings have expanded the application of zebrafish model to study fungal infection. We also proposed a straightforward way to evaluate novel immunomodulation chemicals for their effects in controlling inflammation in gut. With application of automated robotic platform, we expect to make significant progress in understanding pathogenesis of more infectious and inflammatory diseases, and to develop novel therapy for related patients.
Hepatocellular carcinoma (HCC) is a highly invasive malignancy. Recently, GATOR1 (Gap Activity TOward Rags 1) complexes have been shown to play an important role in regulating tumor growth. NPRL2 is ...a critical component of the GATOR1 complex. Therefore, this study used NPRL2 knockdown to investigate how GATORC1 regulates the prognosis and development of HCC via the mammalian target of rapamycin (mTOR) and autophagy signaling pathways. We established HepG2 cells with NPRL2 knockdown using small interfering RNA (siRNA) and short hairpin RNA (shRNA) systems. The siRNA‐mediated and shRNA‐mediated NPRL2 down‐regulation significantly reduced the expression of NPRL2 and two other GATPOR1 complex components, NPRL3 and DEPDC5, in HepG2 cells; furthermore, the efficient down‐regulation of NPRL2 protein expression by both the shRNA and siRNA systems enhanced the proliferation, migration, and colony formation in vitro. Additionally, the NPRL2 down‐regulation significantly increased HCC growth in the subcutaneous and orthotopic xenograft mouse models. The NPRL2 down‐regulation increased the Rag GTPases and mTOR activation and inhibited autophagy in vitro and in vivo. Moreover, the NPRL2 level in the tumors was significantly associated with mortality, recurrence, the serum alpha fetoprotein level, the tumor size, the American Joint Committee on Cancer stage, and the Barcelona Clinic Liver Cancer stage. Low NPRL2, NPRL3, DEPDC5, and LC3, and high p62 and mTOR protein expression in the tumors was significantly associated with disease‐free survival and overall survival in 300 patients with HCC after surgical resection. Conclusion: The efficient down‐regulation of NPRL2 significantly increased HCC proliferation, migration, and colony formation in vitro, and increased HCC growth in vivo. Low NPRL2 protein expression in the tumors was closely correlated with poorer clinical outcomes in patients with HCC. These results provide a mechanistic understanding of HCC and aid the development of treatments for HCC.
Efficient NPRL2 downregulation significantly increased HCC proliferation, migration, and colony formation in vitro, and increased HCC growth in vivo via the mTOR pathway and autophagy suppression. Low NPRL2 protein expression in the tumors was closely correlated with poorer clinical outcomes in HCC patients.
A facile and practical protocol was developed for the synthesis of glycosyl iodides using AlI
3
generated in situ from cheap aluminum metal and molecular iodine. Furthermore, in combination with ...iodine-catalyzed per-O-acetylation, sequential synthesis of per-acetylated glycosyl iodides, per-acetylated thioglycosides, selenoglycoside, and O-glycosides from unprotected reducing sugars was also achieved with complete diastereocontrol in a one-pot version. Supplemental material is available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.
A facile and practical protocol was developed for the synthesis of glycosyl iodides using AlI3 generated in situ from cheap aluminum metal and molecular iodine. Furthermore, in combination with ...iodine-catalyzed per-O-acetylation, sequential synthesis of per-acetylated glycosyl iodides, per-acetylated thioglycosides, selenoglycoside, and O-glycosides from unprotected reducing sugars was also achieved with complete diastereocontrol in a one-pot version. Supplemental material is available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the free supplemental file. PUBLICATION ABSTRACT
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