Jiang, Guozhi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Neuroblastoma is a heterogeneous pediatric malignancy originating from the developing sympathetic nervous system, with poor long-term survival for high-risk patients (~40%). About half of advanced neuroblastomas harbor high-level amplification of the MYCN gene, and these tumors show few, if any, additional driver lesions. Despite significant increase in the body of knowledge of genetics in neuroblastoma, all the high-risk patients follow similar therapeutic procedures and little advancement has been made on molecular target based therapies. The major challenge is to dissect the complexity and heterogeneity of these tumors to find driver genes and activated pathways that are essential for the survival of these cancer cells. We used an integrated systems biology approach to define the core regulatory machinery responsible for maintenance of an aggressive neuroblastoma phenotypic state. In the first part of the thesis, I will discuss our computational approach to decipher the tumor heterogeneity by subtype classification, followed by identification of master regulator protein modules for three distinct molecular subtypes of high-risk neuroblastomas, which were validated in a large independent cohort of cases. We propose that such modules are responsible for integrating the effect of mutations in upstream pathways and for regulating the genetic programs and pathways necessary for tumor state implementation and maintenance. The second part of the thesis is focused on experimental validation of putative master regulators in the subtype of neuroblastomas associated with MYCN amplification. By using RNAi screening followed by experimental and computational analyses to elucidate the interdependencies between the top master regulators, we identified TEAD4-MYCN positive feedback loop as a major tumor maintenance mechanism in this subtype. While MYCN regulates TEAD4 transcriptionally, TEAD4 regulates MYCN through transcriptional and post-translational mechanisms. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally orchestrate 70% of the subtype-specific gene expression signature. TEAD4 gene expression was associated with neuroblastoma patient survival independently of age, tumor stage and MYCN status (P=2.1e-02). In cellular assays, MYCN promoted growth and repressed differentiation, while TEAD4 activated proliferation and DNA damage repair programs, the signature hallmarks of MYCN-amplified neuroblastoma cells. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk neuroblastoma pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. The critical role of the core master regulator module in controlling tumor cell viability, both in vitro and in vivo, and its clinical relevance as a prognostic factor highlights TEAD4 as a novel and highly effective candidate target for therapeutic intervention. In this thesis, we demonstrate that interrogation of tumor specific regulatory networks with patient-derived gene expression signatures can effectively elucidate molecular subtypes as well as the core transcriptional machinery driving subtype specific hallmarks. This approach enables identification of oncogenic and non-oncogenic dependencies of high-risk neuroblastoma and is applicable to other tumor subtypes.

Computational genomics is at the intersection of computational applied physics, math, statistics, computer science and biology. With the advances in sequencing technology, large amounts of comprehensive genomic data are generated every year. However, the nature of genomic data is messy, complex and unstructured; it becomes extremely challenging to explore, analyze and understand the data based on traditional methods. The needs to develop new quantitative methods to analyze large-scale genomics datasets are urgent. By collecting, processing and organizing clean genomics datasets and using these datasets to extract insights and relevant information, we are able to develop novel methods and strategies to address specific genetics questions using the tools of applied mathematics, statistics, and human genetics. This thesis describes genetic and bioinformatics studies focused on utilizing and developing state-of-the-art computational methods and strategies in order to identify and interpret de novo mutations that are likely causing developmental disorders. We performed whole exome sequencing as well as whole genome sequencing on congenital diaphragmatic hernia parents-child trios and identified a new candidate risk gene MYRF. Additionally, we found male and female patients carry a different burden of likely-gene- disrupting mutations, and isolated and complex patients carry different gene expression levels in early development of diaphragm tissues for likely-gene-disrupting mutations. To increase the power to detect risk genes and risk variants, we developed a deep neural network classifier called MVP to accurately predict the pathogenicity of missense variants. MVP implemented an advanced structure of ResNet model and based on two independent data sets, MVP achieved clearly better results in prioritizing pathogenic variants than other methods. Additionally, we studied the genetic connection between developmental disorders and cancer. We found that in developmental disorder patients predicted deleterious de novo mutations are more enriched in cancer driver genes than non cancer driver genes. A Hidden Markov Model was implemented to discover cancer somatic missense mutation hotspots and we demonstrated many cancer driver genes shared a similar mode of action in developmental disorders and caner. By improving ability to interpret missense mutations and leveraging cancer genomics data, we can improve risk gene inference in developmental disorders.

Wang, Xingyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-131). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / 摘要 --- p.VIII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- CLINICAL DISEASE CAUSED BY M.TB --- p.1 / Chapter 1.1.1 --- Tuberculosis (TB) --- p.1 / Chapter 1.1.2 --- Pathogen: Mycobacteria tuberculosis (M. TB) --- p.2 / Chapter 1.2 --- HOST DEFENSE AGAINST M.TB --- p.4 / Chapter 1.2.1 --- Overview --- p.4 / Chapter 1.2.2 --- Specific pathways --- p.6 / Chapter 1.3 --- GENETIC PREDISPOSITION OF HOST TO INFECTION --- p.12 / Chapter CHAPTER 2 --- OVERVIEW AND AIM OF THIS PROJECT --- p.14 / Chapter 2.1 --- GWAS REPLICATION --- p.14 / Chapter 2.2 --- CANDIDATE GENES REVEALED IN GWAS OF OTHER GRANULOMATOUS INFLAMMATORY DISEASES (GLD) --- p.14 / Chapter 2.3 --- CHROMOSOME 17 CHEMOKINE CLUSTER REGION --- p.15 / Chapter CHAPTER 3 --- REPLICATION STUDY OF TB GWAS --- p.16 / Chapter 3.1 --- INTRODUCTION --- p.16 / Chapter 3.1.1 --- TB GWAS study --- p.16 / Chapter 3.1.2 --- Aims of this part --- p.16 / Chapter 3.2 --- MATERIAL AND METHODS --- p.17 / Chapter 3.2.1 --- Case and control samples --- p.17 / Chapter 3.2.2 --- DNA extraction --- p.18 / Chapter 3.2.3 --- Genotyping of the SNPs --- p.19 / Chapter 3.2.4 --- Statistical analysis --- p.21 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Description of studied samples --- p.23 / Chapter 3.3.2 --- Results of case-control study for replication studies of TB GWAS --- p.23 / Chapter 3.4 --- DISCUSSION --- p.28 / Chapter CHAPTER 4 --- GENETIC VARIANTS IN GRANULOMATOUS INFLAMMATORY DISEASES --- p.32 / Chapter 4.1 --- INTRODUCTION --- p.32 / Chapter 4.1.1 --- Granulomatous inflammation --- p.32 / Chapter 4.1.2 --- Diseases characterized by granulomatous inflammatory --- p.34 / Chapter 4.1.3 --- Shared immune mechanisms in GiDs --- p.38 / Chapter 4.1.4 --- Genome-wide Association Studies (GWAS) in GiD --- p.38 / Chapter 4.1.5 --- Hypothesis of this part --- p.41 / Chapter 4.2 --- MATERIAL AND METHODS --- p.43 / Chapter 4.2.1 --- Case and control samples --- p.43 / Chapter 4.2.2 --- DNA extraction --- p.44 / Chapter 4.2.3 --- Tag SNP selection --- p.44 / Chapter 4.2.4 --- Genotyping of tagging SNPs --- p.45 / Chapter 4.2.5 --- Statisitical analysis --- p.45 / Chapter 4.3 --- RESULTS --- p.55 / Chapter 4.3.1 --- Description of TB case samples --- p.55 / Chapter 4.3.2 --- Primary endpoint case-control results --- p.56 / Chapter 4.3.3 --- Secondary endpoint case-only studies results --- p.67 / Chapter 4.3.4 --- Haplotype analysis --- p.78 / Chapter 4.4 --- DISCUSSION --- p.83 / Chapter 4.4.1 --- ATG16L1 gene with TB susceptibility --- p.83 / Chapter 4.4.2 --- Associations in case-only studies (Interaction effects) --- p.83 / Chapter 4.4.2.1 --- Age and pathogenesis of TB --- p.83 / Chapter CHAPTER 5 --- STUDIES IN THE CHEMOKINE-GENE CLUSTER AND A MIRNA SNP STUDY --- p.89 / Chapter 5.1 --- INTRODUCTION --- p.89 / Chapter 5.1.1 --- Genetic susceptibility to TB in familial cases --- p.89 / Chapter 5.1.2 --- Familial studies suggested linkage at 17qll.2 --- p.89 / Chapter 5.1.3 --- Chemokines --- p.90 / Chapter 5.1.4 --- Studies of SNP rs2910164 of microRNA-146a (miRNA-146a) --- p.91 / Chapter 5.2 --- MATERIAL AND METHODS --- p.92 / Chapter 5.2.1 --- Case and control samples --- p.92 / Chapter 5.2.2 --- DNA extraction --- p.92 / Chapter 5.2.3 --- TagSNP selection --- p.92 / Chapter 5.2.4 --- Genotyping of tagging SNPs --- p.93 / Chapter 5.2.5 --- PCR-RFLP --- p.93 / Chapter 5.2.6 --- Statistical analysis --- p.94 / Chapter 5.3 --- RESULTS --- p.100 / Chapter 5.3.1 --- PCR-RFLP results of the three SNPs --- p.100 / Chapter 5.3.2 --- Description of TB case samples --- p.102 / Chapter 5.3.3 --- Primary endpoint case-control results --- p.103 / Chapter 5.3.4 --- Secondary endpoint case-only studies results of CCL genes --- p.109 / Chapter 5.4 --- DISCUSSION --- p.120 / Chapter 5.4.1 --- Genetic association of SNPs with severity of TB --- p.120 / Chapter 5.4.2 --- Smoking and immunity --- p.121 / Chapter CHAPTER 6 --- FINAL CONCLUSION AND PROSPECT FOR FUTURE WORK --- p.122 / Chapter 6.1 --- CONCLUSION --- p.122 / Chapter 6.2 --- LIMITATION OF THE STUDIES --- p.124 / Chapter 6.3 --- FUTURE WORKS AND PROSPECT --- p.125 / REFERENCES --- p.126

Meng, Zhaoyu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 61-74). / Abstracts in English and Chinese. / Declaration of Origination --- p.II / Abstract --- p.III / Acknowledgement --- p.VII / Table of Contents --- p.VIII / List of Tables --- p.XI / List of Figures --- p.XII / List of Abbreviations --- p.XIII / Chapter Chapter 1. --- Review of postoperative pain --- p.1 / Chapter Chapter 2. --- Candidate Genes --- p.6 / Chapter 2.1 --- μ Opioid Receptor Gene --- p.6 / Chapter 2.2 --- ATP-binding cassette B1 (ABCB1) / multiple drug resistance 1 (MDR1) gene --- p.10 / Chapter 2.3 --- Catechol-O-methyltransferase (COMT) gene --- p.10 / Chapter 2.4 --- βArrestin2 gene --- p.11 / Chapter 2.5 --- Transient receptor potential (TRP) gene --- p.11 / Chapter 2.6 --- GTP cyclohydrolase 1 (GCHl)gene --- p.12 / Chapter Chapter 3. --- Hypothesis and Experimental Design --- p.15 / Chapter 3.1 --- Hypothesis --- p.15 / Chapter 3.2 --- Objectives --- p.15 / Chapter 3.3 --- Experimental design --- p.15 / Chapter 4.4 --- Role in this study --- p.16 / Chapter Chapter 4. --- Methods --- p.17 / Chapter 4.1 --- Patients --- p.17 / Chapter 4.2 --- Anesthetic procedure --- p.17 / Chapter 4.3 --- Postoperative management --- p.18 / Chapter 4.4 --- Patient follow-up --- p.19 / Chapter 4.5 --- Definition of endpoints --- p.19 / Chapter Chapter 5. --- Genotyping --- p.22 / Chapter Chapter 6 --- Statistical Analysis and Sample Size --- p.24 / Chapter 6.1 --- Statistical analysis --- p.24 / Chapter 6.2 --- Sample size --- p.26 / Chapter Chapter 7. --- "Demographic, genotype and haplotype result" --- p.28 / Chapter 7.1 --- Patient characteristics --- p.28 / Chapter 7.2 --- Genotypes and haplotypes --- p.30 / Chapter Chapter 8. --- Acute postoperative pain --- p.35 / Chapter Chapter 9. --- Morphine consumption for early postoperative pain --- p.41 / Chapter 9.1 --- Patients using alternative analgesia --- p.41 / Chapter 9.2 --- Result in the patients using morphine alone --- p.41 / Chapter Chapter 10. --- Opioid-related Side Effects --- p.47 / Chapter Chapter 11. --- Chronic postoperative pain --- p.49 / Chapter 11.1 --- Patient characteristics --- p.49 / Chapter 11.2 --- Association of genetic polymorphisms and Chronic postoperative pain --- p.51 / Chapter Chapter 12. --- Discussion --- p.54 / Chapter 12.1 --- Acute postoperative pain --- p.54 / Chapter 12.2 --- Chronic postoperative pain --- p.57 / Chapter Chapter 13. --- Conclusion --- p.60 / References --- p.61 / Appendix I. Morphine Consumption and and Number of Demands for Patient Controlled Analgesia (PCA) in Patients with Different Genotypes --- p.75 / Appendix II. Opioid Related Symptom Distress Scale Score on Day 1 and Overall Score of Each Day in Patients with Different Genotypes --- p.79 / Appendix III. Opioid Related Symptoms Distress Scale Score in Patients with Different Genotypes --- p.83

Single nucleotides in the first intron of FTO convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes (e.g. RPGRIP1L, IRX3) in the vicinity of FTO. This locus affects food intake, the browning of white adipose tissue and risk of type 2 diabetes (independent of its effects on body weight). FTO and RPGRIP1L expression are decreased in fibroblasts and iPSC-derived human neurons of individuals segregating for obesity risk alleles of FTO at rs8050136 and rs1421085. These alleles exhibit decreased binding of isoform p110 of the CUX1 transcription factor. This isoform activates transcription of both FTO and RPGRIP1L. The FTO locus conveys effects on adiposity via hyperphagia, in part, by regulating FTO and RPGRIP1L expression in the hypothalamus. We examined whether FTO and RPGRIP1L also modify adipogenesis and adipose tissue lipid storage. Such effects would influence systemic consequences of the hyperphagia driven by the actions of the genes in the hypothalamus. Given the role in energy homeostasis of genes encoding elements of the primary cilium, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. In confirmation, we find that Rpgrip1l+/− mice are hyperphagic and obese, and display diminished suppression of food intake in response to leptin administration. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO intronic locus. We describe effects of Rpgrip1l in adipocytes which may contribute to the adiposity phenotype observed in these animals, and possibly humans. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes (using Adipoq-Cre) did not increase adiposity in mice fed chow or high fat diet. Neither did we observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes in vitro. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it appears to do so by effects conveyed in preadipocytes, a cell type in which the primary cilium – as a mediator of developmental signals – may have functional importance. We propose that decreased RPGRIP1L expression in preadipocytes in humans segregating for FTO-associated obesity risk alleles increases the potential storage capacity of adipose tissue. Such capacity would influence the metabolic consequences of positive energy balance due to the action of these alleles within the brain. Fto expression is upregulated during adipogenesis in murine and human cells in vitro, and is more highly expressed in isolated mouse adipocytes than in preadipocytes. Here we demonstrate that FTO is required for the maintenance of adipocyte lipid filling and endocrine function in murine 3T3-L1 cells and human adipose tissue-derived stromal cells. RNAseq analysis indicates that this effect on adipocyte programming is conveyed in part by modulation of C/ebpβ- and C/ebpδ-regulated transcription, consistent with reports that Fto acts a transcriptional coactivator. Fto-/- mice have normal fat mass in early life, but spontaneously lose adipose tissue as they age. We propose that Fto is required to maintain adipocyte viability, a function critical to the prevention of ectopic lipid accumulation in obese states. Such accumulation – both total and in specific anatomic regions – has adverse metabolic consequences. In addition to the developmental effects on adiposity mediated by RPGRIP1L, and the effects conveyed on adipocyte function related to FTO, the FTO locus could also impact systemic energy homeostasis by modifying production of humoral signals that are integrated centrally to regulate energy balance. We explored molecular modifiers of adipocyte production of leptin identified by GWAS that may modify obesity risk. The FTO locus was associated with circulating leptin concentration, but this association was abrogated when corrected for BMI, indicating that this locus does not contribute to adiposity by dysregulating leptin production. Our in vitro findings are consistent in this regard, as knockdown of Rpgrip1l and Fto in 3T3-L1 cells did not affect leptin production per adipocyte. These results, however, are not inconsistent with a role for FTO in maintenance of adipocyte viability.

Sze, Siu Ching. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 109-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

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