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Obstructive sleep apnea syndrome (OSAS) is a complex disorder characterized by repetitive bouts of upper airway collapse during sleep, causing subsequent intermittent hypoxia, hypercapnia, and fragmented sleep and is also associated with significant morbidity including daytime sleepiness, hypertension, and elevated cardiovascular risk. OSAS affects at least 4% of men and 2% of women; unfortunately, it is estimated that 80% to 90% of adults with OSAS remain undiagnosed. Both clinical characteristics and complex genetic and environmental interactions have made it difficult to understand OSAS disease etiology and identifying patients at risk is still elusive. A pattern of gene expression in cells or tissues related to a disease state for OSAS would provide beneficial information to be most effective in screening or diagnosing this disease. Objectives: The objectives of this study were to: 1) map out the study design and bench assay strategies by which to investigate this issue; 2) find out if there are specific differences in the global gene expression profiles of adult females with OSAS compared to those without OSAS, under conditions in which subjects were clinically similar (BMI, diabetes, cardiovascular disease, etc.); and 3) assess the protein expression differences that could potentially be linked via well-established molecular pathways associated with any differences found in global gene expression profiles in the presence and absence of OSAS. Methods: Subjects were overweight premenopausal Caucasian women with untreated OSAS (n=6; age = 40.7 ± 3.4; BMI = 49.04 ± 6.97; apnea-hypopnea index = 27.3 ± 16.02), and control subjects (n=10) (age = 38.2 ± 7.6; BMI = 47.94 ± 6.15; apnea-hypopnea index < 5), and matched for other clinical characteristics (diabetes, cardiovascular disease status, medications, etc.) recruited from either Carilion Clinic Pulmonary/Sleep Medicine or Carilion Clinic Bariatric Surgery practices. Subjects provided a fasting blood sample in which the monocytes were isolated from whole blood. The RNA was extracted from the monocytes, assessed for purity and quantity, frozen and shipped to collaborators at Dana-Farber Cancer Institute and hybridized to Affymetrix whole human genome chips on a gene chip. The initial computational evaluation and interpretation generated the hypothesis. Two-step quantitative real time polymerase chain reaction (qPCR) was performed to verify the results from the microarray analysis. The laminin enzyme immunoassay (EIA), and cellular adhesion assays were performed to determine if genomic changes resulted in proteomic and phenotypic assessments. Results: OSAS subjects had nine aberrantly regulated genes, of which three genes (LAMC-1, CDC42, and TACSTD2) showed a pattern in segregation between OSAS and controls subjects based on expression patterns. In addition, qPCR indicated a 2.1 fold increase in LAMC-1 and a 1.1 fold increase CDC42 expression unique to the tissue samples of patients with OSAS. Though the serum laminin EIA did not differ between groups, a statistically significant increase in peripheral blood mononuclear cells (PBMC) cellular adhesion in OSAS patients versus control subjects was found. The OSAS subjects had a well cell count of 9.27 ± 1.54 cells vs. controls 5.75 ± 0.78 cells (p Ë‚ 0.05), which is relative to the 103 cells/field that were plated. Conclusions: Cells isolated from women with moderate-severe OSAS show an abnormality in cellular adhesion, a process driven in part by the gene LAMC-1, which was also aberrantly expressed in these subjects. This suggests that inflammation may be linked to the pathogenesis of OSAS. This pilot study has provided the framework and preliminary data needed to propose a larger study with extramural research funding. / Ph. D.

Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance. cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs. Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that regulate gene expression at both the mRNA and protein levels. MiRNAs have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. We investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N =35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain-specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. Given that miRNAs can bind to their targets with imperfect complementarity, computational prediction of true miRNA:mRNA interactions has been difficult and therefore, functional validation of miRNA:mRNA interactions has been relatively sparse. Thus, it was the goal of this study to demonstrate biological functionality of miRNAs on their targets by evaluating transcriptional and translational levels of gene expression(real-time PCR, western blot) as well as determining miRNA target-site specificity (luciferase reporter gene assays). We investigated two miRNAs, miR-132 and miR-137, both of which have been shown to regulate neuronal function and development, and are believed to be associated with schizophrenia from two distinct avenues of research, miR-132 from expression studies and miR-137 from genetic studies. We demonstrated miR-132 down-regulates NTF3, DISC1, and GRIK5 at the transcript level and down-regulates GRIK5 at the protein level as well. Furthermore, we demonstrated miR-137 down-regulates TCF4, CACNA1C, CDK6, ANK3, and ZNF804A at the transcript level, and down-regulates TCF4, CACNA1C, and CDK6 at the protein level. Going further, we also demonstrated miR-137 binds specifically to target sites in the 3'-UTR of CACNA1C, TCF4, and CDK6, suggesting repression of these genes is directly mediated by miR-137. In total, this study provides strong evidence that miRNA dysregulation may contribute to schizophrenia pathogenesis.

Le bulbe olfactif (BO) des mammifères adultes est le siège d'une intense neurogenèse tout au long de la vie. L'intégration des nouveaux neurones dans le BO est alimentée par la génération continuelle de progéniteurs immatures dans la zone periventriculaire (ZPV) du ventricule latéral du cerveau antérieur. Au cours de leur différentiation, ceux-ci migrent « en chaine » de la ZPV vers le BO. Une fois dans le BO ils migrent alors radialement vers leur localisation finale et achèvent leur différentiation. Le phénotype des neurones néoformés est divers et est déterminé par la position des cellules souche dans la ZPV. Outre un intérêt spécifique, cette neurogenèse offre des perspectives uniques pour étudier la neurogenèse en général. En effet, dans ce système, les étapes successives du processus de différentiation sont distinctement séparées dans l'espace.Durant ma thèse j'ai étudié le rôle des microARN dans la neurogenèse du BO. Les microARN sont des ARN d'environs 22 nucléotides qui régulent négativement l'expression des gènes au niveau post-transcriptionnel. En utilisant des souris mutantes conditionnelles pour une enzyme clé dans la synthèse des microARN, j'ai démontré que les microARN étaient essentiels à la génération de nouveaux neurones. Par la suite, pour identifier des microARN candidats, le profil d'expression de l'ensemble des microARN durant la neurogenèse a été réalisé. Cette étude s'est faite par séquençage haut-débit des petits ARN sur un panel d'échantillons représentatifs des différentes étapes de la neurogenèse du BO et des différents compartiments de cellules souche de la ZPV. / New neurons are continuously and extensively generated in the adult mammalian olfactory bulb (OB). The constant integration of new neurons into the OB circuitry is fueled by the continuous generation of immature progenitors in the periventricular zone (PVZ) of the lateral ventricle of the forebrain. Immature precursor cells leave the PVZ and migrate in interwoven chains to the OB. After arrival in the OB they migrate radially to their final positions and undergo terminal differentiation. The phenotype of these new neurons is diverse and determined by the position of the stem cells in the PVZ. Beyond its specific interest, this system of postnatal neurogenesis provides unique, advantageous properties to study neurogenesis in general, as the distinct steps of the neurogenic sequence (stem cell, amplification, migration, final differentiation) are clearly spatially separated. During my PhD I aimed to elucidate the roles of microRNA mediated regulation of gene expression in the OB neurogenesis. MicroRNAs are a class of small regulatory RNAs around 22 nucleotides in length. They act as negative regulators of gene expression on a post-transcriptional level thereby restricting protein output. Using a conditional knock-out mouse line for a key enzyme of microRNAs synthesis, I first demonstrated that microRNAs are absolutely required to complete the neuronal differentiation process. Subsequently, in order to identify candidates playing a role in neurogenesis, a miRNome profiling was performed by deep sequencing of small RNAs in tissues representative for different stem cell compartments and steps of neurogenesis.

Ng Yeung Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 180-209). / Abstracts in English and Chinese. / acknowledgements --- p.i / abstract --- p.ii / abstract in chinese --- p.vi / table of contents --- p.ix / list of tables --- p.xiii / list of figures --- p.xi v / list of abbreviations --- p.xvi / Chapter 1 --- chapter1 introduction and literature review --- p.1 / Chapter 1.1 --- Introduction of brain tumors --- p.1 / Chapter 1.2 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.2.1 --- Oligodendroglioma (OD) and anaplastic oligodendroglioma (AOD) --- p.3 / Chapter 1.2.1.1 --- WHO's definition and grading --- p.3 / Chapter 1.2.1.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.3 / Chapter 1.2.1.3 --- Clinical presentation --- p.4 / Chapter 1.2.1.4 --- Macroscopy and histopathology --- p.4 / Chapter 1.2.1.5 --- Immunohistochemistry --- p.5 / Chapter 1.2.1.6 --- Treatment --- p.6 / Chapter 1.2.2 --- Oligoastrocytoma (OA) and anaplastic oligoastrocytoma (AOA) --- p.11 / Chapter 1.2.2.1 --- WHO's definition and grading --- p.11 / Chapter 1.2.2.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.12 / Chapter 1.2.2.3 --- Clinical features --- p.12 / Chapter 1.2.2.4 --- Macroscopy and histopathology --- p.12 / Chapter 1.3 --- Overview of Genetic and Epigenetic Aberrations of OTs --- p.14 / Chapter 1.3.1 --- Chromosomal and genetic aberrations in OTs --- p.14 / Chapter 1.3.2 --- Candidate regions and genes on 1 p --- p.15 / Chapter 1.3.3 --- Candidate regions and genes on 19q --- p.20 / Chapter 1.3.4 --- Other aberrations in WHO grade II OTs --- p.24 / Chapter 1.3.5 --- Progression-associated aberrations in ODs --- p.25 / Chapter 1.3.6 --- Chromosomal and genetic aberrations in OAs --- p.29 / Chapter 1.4 --- Correlation of genetic alterations with response to therapy and survival --- p.31 / Chapter 1.4.1 --- Response to PCV chemotherapy correlates with lp and combined lp/19q status in patients with AODs --- p.31 / Chapter 1.4.2 --- Survival of patients with AODs correlates with lp/19q status --- p.32 / Chapter 1.4.3 --- WHO grade II ODs behavior and lp/19q status --- p.32 / Chapter 1.4.4 --- Response to other therapies (temozolomide and radiotherapy) and lp/19q status in patients with ODs --- p.33 / Chapter 1.4.5 --- lp and 19q loss in OAs and diffuse astrocytomas --- p.34 / Chapter 1.5 --- Microarray-based expression profiling of OTs --- p.35 / Chapter 1.6 --- Description of p73 protein --- p.37 / Chapter 1.6.1 --- Introduction of p73 --- p.37 / Chapter 1.6.2 --- p73: gene structure and splicing variants --- p.37 / Chapter 1.6.3 --- Signaling in p73 --- p.40 / Chapter 1.6.4 --- Regulation ofp73 protein stability and transcriptional activity --- p.43 / Chapter 1.6.4.1 --- Regulation by DNA damage --- p.43 / Chapter 1.6.4.2 --- Regulation by oncogenes --- p.44 / Chapter 1.6.4.3 --- Interaction with viral proteins --- p.44 / Chapter 1.6.5 --- Role of p73 in the nervous system --- p.45 / Chapter 1.6.6 --- p73 in cancer --- p.45 / Chapter 1.6.6.1 --- p73 knockout mice --- p.45 / Chapter 1.6.6.2 --- Alteration of p73 expression in human cancers --- p.46 / Chapter 1.6.7 --- p73 and chemosensitivity --- p.50 / Chapter CHAPTER2 --- AIMS OF STUDY --- p.51 / Chapter CHAPTER3 --- MATERIALS AND METHODS --- p.53 / Chapter 3.1 --- Tumor and blood samples --- p.53 / Chapter 3.2 --- Cell culture --- p.53 / Chapter 3.3 --- DNA extraction from frozen tissues and blood samples --- p.54 / Chapter 3.4 --- Detection of allelic loss of chromosome lp --- p.58 / Chapter 3.4.1 --- LOH analysis --- p.58 / Chapter 3.4.2 --- Fluorescence in situ Hybridization (FISH) analysis on Paraffin and Frozen Sections --- p.60 / Chapter 3.6 --- DNA sequencing analysis --- p.62 / Chapter 3.7 --- Analysis of Methylation --- p.63 / Chapter 3.7.1 --- Bisulfite sequencing --- p.63 / Chapter 3.7.2 --- Methylation-specific polymerase chain reaction (MSP) --- p.66 / Chapter 3.8 --- Northern Blot analysis --- p.68 / Chapter 3.9 --- RNA isolation and cDNA preparation --- p.70 / Chapter 3.10 --- Laser microdissection and RNA extraction from microdissected tumor cells --- p.71 / Chapter 3.10.1 --- Conventional RT-PCR --- p.71 / Chapter 3.11 --- Primer design for TP73 and its isoforms --- p.74 / Chapter 3.12 --- Real-time RT-PCR --- p.77 / Chapter 3.12.1 --- Real-time RT-PCR for TP73 and its isoforms --- p.78 / Chapter 3.12.2 --- Real-time RT-PCR for KIAA0495 --- p.79 / Chapter 3.13 --- Statistical analyses --- p.81 / Chapter CHAPTER4 --- RESULTS --- p.82 / Chapter 4.1 --- Genes annotated in the minimally deleted regions --- p.82 / Chapter 4.2 --- Expression analyses of TP73 and its isoforms in ODs by quantitative real-time RT-PCR --- p.85 / Chapter 4.3 --- Methylation analysis of TP73 in ODs by methylation sensitive PCR (MSP) --- p.97 / Chapter 4.4 --- A rapid screen of candidate genes for aberrant expression in microdissected tumors --- p.100 / Chapter 4.5 --- Quantitative real-time RT-PCR of KIAA0495 gene --- p.103 / Chapter 4.6 --- Mutation analysis of KIAA0495 gene --- p.110 / Chapter 4.7 --- Methylation analysis of KIAA0495 in ODs by bisulfite sequencing…… --- p.112 / Chapter 4.8 --- Detection of allelic loss of lp by LOH analysis and interphase FISH --- p.121 / Chapter 4.9 --- Two-hit inactivation of KIAA0495 gene in ODs --- p.126 / Chapter 4.10 --- Tissue distribution of KIAA0495 gene --- p.130 / Chapter 4.11 --- Bioinformatics of KIAA0495 --- p.133 / Chapter CHAPTER5 --- DISCUSSION --- p.146 / Chapter 5.1 --- Expression analysis of TP73 and its isoforms in ODs by isoform-specific RT-PCR --- p.148 / Chapter 5.2 --- Methylation status ofTP73 in ODs --- p.153 / Chapter 5.3 --- A rapid screening of candidate genes for aberrant expressionin microdissected tumors --- p.156 / Chapter 5.4 --- Expression pattern of KIAA0495 mRNA in a large cohort of ODs --- p.157 / Chapter 5.5 --- No somatic mutation in coding region of KIAA0495 --- p.158 / Chapter 5.6 --- Methylation status of putative promoter region of KIAA0495 in ODs --- p.159 / Chapter 5.7 --- Status of chromosome lp in ODs --- p.161 / Chapter 5.8 --- Two-hit inactivation of KIAA0495 gene in ODs by promoter hypermethylation and allelic loss of lp --- p.162 / Chapter 5.9 --- Evaluation of expression of KIAA0495 gene as a marker for the response to chemotherapy and prognostic marker in patients with OTs --- p.164 / Chapter 5.10 --- Tissue distribution of KIAA0495 --- p.166 / Chapter 5.11 --- "KIAA0495 cDNA sequence, protein sequence and potential functional features" --- p.167 / Chapter 5.12 --- Candidate tumor suppressor genes on lp in other type of tumors with loss of lp --- p.171 / Chapter CHAPTER6 --- CONCLUSIONS --- p.174 / Chapter CHAPTER7 --- FUTURE STUDIES --- p.177 / Chapter CHAPTER8 --- REFERENCES --- p.180

Yu, Pui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 68-78). / Abstracts in English and Chinese. / Acknowledgements --- p.iv / Abstract --- p.v / Table of Contents --- p.ix / List of Figures --- p.xii / List of Tables --- p.xiv / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.5 / Chapter 2.1 --- The importance of terpenoids --- p.5 / Chapter 2.2 --- The difficulties in synthesizing terpenoids --- p.8 / Chapter 2.3 --- Structure and classification of terpenoids --- p.9 / Chapter 2.4 --- MVA and MEP pathways of terpenoid biosynthesis in higher plants --- p.12 / Chapter 2.4.1 --- The MVA pathway --- p.16 / Chapter 2.4.2 --- The MEP pathway --- p.18 / Chapter 2.5 --- The crosstalk between MVA and MEP routes --- p.20 / Chapter 2.6 --- The farnesyl diphosphate is a key enzyme in terpenoid biosynthetic pathway --- p.20 / Chapter 2.7 --- The glutaredoxin system --- p.22 / Chapter Chapter 3. --- Materials and Methods --- p.25 / Chapter 3.1 --- Plant materials and growth condition --- p.25 / Chapter 3.2 --- DNA extraction and screening of fps mutants --- p.25 / Chapter 3.3 --- Validation of the fps mutant by semi-quantitative RT-PCR --- p.26 / Chapter 3.4 --- Semi-quantitative RT-PCR analysis of the fps mutants --- p.28 / Chapter 3.5 --- Genechip analysis of fps mutants --- p.29 / Chapter 3.6 --- Enzyme assays --- p.29 / Chapter 3.7 --- Triterpene and sterol analysis of fps mutants --- p.30 / Chapter 3.8 --- Preparation of carotenoid standards for carotenoid analysis --- p.31 / Chapter 3.9 --- Carotenoids analysis of fps mutants by HPLC --- p.31 / Chapter 3.10 --- Subcellular localization of FPS 1 and FPS2 by transient expression --- p.33 / Chapter Chapter 4. --- Results --- p.36 / Chapter 4.1 --- Screening of fpsl and fps2 homozygous mutants --- p.36 / Chapter 4.2 --- Validation of fps mutants by RT-PCR and enzyme activity assay --- p.36 / Chapter 4.3 --- Genechip analysis of two fps mutants --- p.40 / Chapter 4.3.1 --- Quality control and normalization of microarray sample --- p.40 / Chapter 4.3.2 --- Normalization and identification of differentially expressed genes --- p.42 / Chapter 4.3.3 --- GO annotation of differentially expressed genes in fps mutants --- p.43 / Chapter 4.3.4 --- Genes participate in stress and defense response were differentially expressed in both fpsl and fps2 mutants --- p.48 / Chapter 4.3.5 --- Genes in the plastidial pathway were down-regulated --- p.51 / Chapter 4.4 --- Effects of FPS mutations on pathway enzymes --- p.53 / Chapter 4.5 --- Effects of fps mutants on terpenoids and sterol metabolism --- p.55 / Chapter 4.6 --- Comparison on carotenoids and chlorophyll contents --- p.55 / Chapter 4.7 --- Subcellular localization of FPS 1 and FPS2 --- p.61 / Chapter Chapter 5. --- Discussion --- p.62 / Chapter Chapter 6. --- Conclusion --- p.67 / Reference --- p.68 / Appendices --- p.79 / Appendix A. Primers designed for homozygous mutant screening for fps mutants --- p.79 / "Appendix B. Primer pairs designed for fps ORF, common sequence and fpsl specific region" --- p.80 / Appendix C. Primer pairs designed for studying expression level of the downstream genes of FPS --- p.81 / Appendix D. Annotations of differentially expressed genes in fpsl mutant --- p.82 / Appendix E. Annotations of differentially expressed genes in fps2 mutant --- p.84 / Appendix F. Log fold changes of terpenoid pathway genes involved in FPS mutants --- p.94

Qi, Yan. / Thesis submitted in: December 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 63-79). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.III / ABSTRACT --- p.IV / TABLE OF CONTENTS --- p.VII / LIST OF ABBREVIATIONS --- p.XI / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- the Plant of Artemisia annua L --- p.1 / Chapter 1.2 --- The disease of malaria --- p.3 / Chapter 1.2.1 --- The life cycle of Plasmodium parasites --- p.4 / Chapter 1.2.2 --- The Artemisinin-based combination therapies (ACTs) for the treatment of malaria --- p.5 / Chapter 1.3 --- Artemisinin --- p.8 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.8 / Chapter 1.3.2 --- The mechanism of artemisinin action --- p.9 / Chapter 1.3.2.1 --- The proposed non-specific mechanisms of action --- p.10 / Chapter 1.3.2.2 --- The proposed parasite-specific mechanisms of action --- p.11 / Chapter 1.3.3 --- The biosynthesis of artemisnin in vivo --- p.12 / Chapter 1.3.4 --- The biosynthesis of artemisinin in vitro --- p.16 / Chapter 1.4 --- Trichomes --- p.18 / Chapter 1.4.1 --- Non-glandular trichomes --- p.19 / Chapter 1.4.2 --- Glandular trichome --- p.20 / Chapter 1.4.3 --- Trichomes of Artemisia annua L --- p.21 / Chapter 1.5 --- DNA Sequencing Methods --- p.24 / Chapter 1.5.1 --- The basic principle of pyrosequencing --- p.25 / Chapter 1.5.2 --- 454 pyrosequencing and its application --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Chemicals --- p.32 / Chapter 2.2 --- Plant materials --- p.32 / Chapter 2.3 --- Preparation of the cDNA sample for 454 sequencing --- p.33 / Chapter 2.3.1 --- Scanning electron microscopy --- p.33 / Chapter 2.3.2 --- Isolation of glandular trichomes --- p.34 / Chapter 2.3.3 --- cDNA synthesis and normalization --- p.34 / Chapter 2.4 --- 454-EST SEQUENCING AND PROCESSING --- p.36 / Chapter 2.5 --- Analysis of 454 sequencing data --- p.37 / Chapter 2.6 --- Establishment of regeneration system of A. annua L --- p.37 / Chapter 2.6.1 --- Shoots induction from leaf discs --- p.37 / Chapter 2.6.2 --- The sensitivity of the explants to Kanamycin --- p.38 / Chapter 2.6.3 --- Rooting of the regenerated seedlings --- p.38 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.40 / Chapter 3.1 --- Glandular trichome isolation and cDNA preparation --- p.40 / Chapter 3.1.1 --- The distribution of glandular trichomes on A. annua --- p.40 / Chapter 3.1.2 --- The isolation of glandular trichomes --- p.42 / Chapter 3.1.3 --- The preparation of ds cDNA for 454 sequencing --- p.43 / Chapter 3.2 --- Pre-process of 454 pyrosequencing data --- p.44 / Chapter 3.3 --- Functional annotation of the 454-EST data --- p.47 / Chapter 3.4 --- Comparison of two sequencing runs --- p.49 / Chapter 3.5 --- Analysis of the 454 ESTs involved in secondary metabolisms --- p.50 / Chapter 3.6 --- Selection of the candidate genes --- p.55 / Chapter 3.7 --- Establishment of regeneration system of A. annua L --- p.57 / Chapter 3.7.1 --- Shoots induction from leaf discs --- p.57 / Chapter 3.7.2 --- Roots induction from shoots --- p.57 / Chapter 3.7.3 --- Sensitivity of A. annua to Kan --- p.59 / Chapter CHAPTER 4. --- CONCLUSION --- p.61 / REFERENCES --- p.63

高通量大規模並行測序技術，又称為二代測序（NGS），極大的加速了生物和醫學研究的進程。隨著測序通量和複雜度的不斷提高，在分析大量的資料以挖掘其中的資訊的過程中，生物訊息學變得越發重要。在我的博士研究生期間（及本論文中），我主要從事於以下兩個領域的生物訊息學演算法的開發：DNA甲基化資料分析和基因間區長鏈非編碼蛋白RNA（lincRNA）的鑒定。目前二代測序技術在這兩個領域的研究中有著廣泛的應用，同時急需有效的資料處理方法來分析對應的資料。 / DNA甲基化是一種重要的表觀遺傳修飾，主要用來調控基因的表達。目前，全基因組重亞硫酸鹽測序（BS-seq）是最準確的研究DNA甲基化的實驗方法之一，該技術的一大特點就是可以精確到單個堿基的解析度。為了分析BS-seq產生的大量測序數據，我參與開發並深度優化了Methy-Pipe軟體。Methy-Pipe集成了測序序列比對和甲基化程度分析，是一個一體化的DNA甲基化資料分析工具。另外，在Methy-Pipe的基礎上，我又開發了一個新的用於檢測DNA甲基化差異區域（DMR）的演算法，可以用於大範圍的尋找DNA甲基化標記。Methy-Pipe在我們實驗室的DNA甲基化研究項目中得到廣泛的應用，其中包括基於血漿的無創產前診斷（NIPD）和癌症的檢測。 / 基因間區長鏈非編碼蛋白RNA（lincRNA）是一種重要的調節子，其在很多生物學過程中發揮作用，例如轉錄後調控，RNA的剪接，細胞老化等。lincRNA的表達具有很強的組織特異性，因此很大一部分lincRNA還沒有被發現。最近，全轉錄組測序技術（RNA-seq）結合基因從頭組裝，為新的lincRNA鑒定以及構建完整的轉錄組列表提供了最有力的方法。然而，有效並準確的從大量的RNA-seq測序數據中鑒定出真實的新的lincRNA仍然具有很大的挑戰性。為此，我開發了兩個生物訊息學工具：1）iSeeRNA，用於區分lincRNA和編碼蛋白RNA（mRNA）；2）sebnif，用於深層次資料篩選以得到高品質的lincRNA列表。這兩個工具已經在多個生物學系統中使用並表現出很好的效果。 / 總的來說，我開發了一些生物訊息學方法，這些方法可以幫助研究人員更好的利用二代測序技術來挖掘大量的測序數據背後的生物學本質，尤其是DNA甲基化和轉錄組的研究。 / High-throughput massive parallel sequencing technologies, or Next-Generation Sequencing (NGS) technologies, have greatly accelerated biological and medical research. With the ever-growing throughput and complexity of the NGS technologies, bioinformatics methods and tools are urgently needed for analyzing the large amount of data and discovering the meaningful information behind. In this thesis, I mainly worked on developing bioinformatics algorithms for two research fields: DNA methylation data analysis and large intergenic noncoding RNA discovery, where the NGS technologies are in-depth employed and novel bioinformatics algorithms are highly needed. / DNA methylation is one of the important epigenetic modifications to control the transcriptional regulations of the genes. Whole genome bisulfite sequencing (BS-seq) is one of the most precise methodologies for DNA methylation study which allows us to perform whole methylome research at single-base resolution. To analyze the large amount of data generated by BS-seq experiments, I have co-developed and optimized Methy-Pipe, an integrated bioinformatics pipeline which can perform both sequencing read alignment and methylation state decoding. Furthermore, I’ve developed a novel algorithm for Differentially Methylated Regions (DMR) mining, which can be used for large scale methylation marker discovery. Methy-Pipehas been routinely used in our laboratory for methylomic studies, including non-invasive prenatal diagnosis and early cancer detections in human plasma. / Large intergenic noncoding RNAs, or lincRNAs, is avery important novel family of gene regulators in many biological processes, such as post-transcriptional regulation, splicing and aging. Due to high tissue-specific expression pattern of the lincRNAs, a large proportion is still undiscovered. The development of Whole Transcriptome Shotgun Sequencing, also known as RNA-seq, combined with de novo or ab initio assembly, promises quantity discovery of novel lincRNAs hence building the complete transcriptome catalog. However, to efficiently and accurately identify the novel lincRNAs from the large transcriptome data stillremains a bioinformatics challenge.To fill this gap, I have developed two bioinformatics tools: I) iSeeRNAfor distinguishing lincRNAs from mRNAs and II) sebnif for comprehensive filtering towards high quality lincRNA screening which has been used in various biological systems and showed satisfactory performance. / In summary, I have developed several bioinformatics algorithms which help the researchers to take advantage of the strength of the NGS technologies(methylome and transcriptome studies) and explore the biological nature behind the large amount of data. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Kun. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 118-126). / Abstracts also in Chinese.

Apomixis, asexual or clonal seed production in plants, can decrease the cost of producing hybrid seed and enable currently open pollinated crops to be converted to more vigorous and higher yielding hybrids that can reproduce themselves through their own seed. Sexual reproduction may be triggered by a programmed stress signaling event that occurs in both the meiocyte, just prior to meiosis, and later in the egg just prior to embryo sac maturation. The prevention of stress signaling and the activation of a pro-growth signal prior to meiosis triggered apomeiosis, the first half of apomixis. The same approach was used prior to embryo sac maturation to trigger parthenogenesis, the second half of apomixis. This discovery suggests that apomixis exists as a program that can be activated by the appropriate metabolic signal at the appropriate developmental stages. Therefore, apomixis may be alternative mode of reproduction rather a ‘broken’ form of sexual reproduction.