Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance. By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila. How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs. The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.

Made available in DSpace on 2016-06-24T04:00:22Z (GMT). No. of bitstreams: 1 Sergio Araujo Figueiredo.pdf: 5051471 bytes, checksum: 845523d46a114f99e92e4c1f07b80e2a (MD5) Previous issue date: 2011-06-03 / ß-galactosidases are a class of glycosyl-hydrolases that act on the plant cell primary walls, hydrolyzing ß-D-galactose at the nonreducing ends of ß-D-galactosides present in several biological molecules. Initially a characterization of the monosaccharides present in the primary wall of the pericarp and endosperm of Coffea arabica fruits at different ripening stages was performed, identifying the polysaccharides present in these regions, along with the putative carbohydrate target for the ß-galactosidase. In parallel, a molecular and a biochemical characterization of ß-galactosidase was performed. A partial characterization of ß-galactosidase genomic DNA structure, along with a transcription analysis and an in vitro and in situ biochemical activity were performed, identifying peaks of expression in the early stages of growth and in fully ripe fruit. Finally, in order to evaluate the ß-galactosidase effects on coffee fruit ripening, C. arabica calli were transformed by biolistic using RNA interference approach, in order to obtain genetically modified coffee plants with a silenced ß-galactosidase expression. Three transgenic calli growing on selective medium containing ammonium glufosinate were obtained, two of which contained the ß-galactosidase gene fragment. These calli are under embryogenic regeneration and the resulting seedlings will be further analyzed in order to confirm the presence of the transgenes and to assess of the effects of ß-galactosidase gene silencing on coffee fruit ripening. / As ß-galactosidases sao uma classe de glicosil-hidrolases que atuam na parede primaria das celulas vegetais, hidrolisando residuos ß-D-galactosis de extremidades nao redutoras de ß-D galactosideos presentes em diversas moleculas biologicas. Inicialmente foi feita uma caracterizacao dos monossacarideos presentes na parede primaria do pericarpo e do endosperma de frutos de Coffea arabica em distintas fases de maturacao, identificando os polissacarideos presentes nesta regiao celular, juntamente com os provaveis carboidratos-alvo para as ß-galactosidases. Em paralelo, foi realizada uma caracterizacao molecular e bioquimica das ß-galactosidases. Foi realizada uma caracterização parcial da estrutura do seu DNA genomico, juntamente com uma analise do nivel de transcricao e da atividade bioquimica in vitro e in situ foram realizadas, identificando picos de expressao nas fases iniciais de crescimento e nos frutos completamente maduros. Por fim, visando avaliar os efeitos das ß-galactosidases na maturacao de frutos de cafe, calos embriogenicos de C. arabica foram transformados por biobalistica, utilizando a tecnica do RNA interferente, com a finalidade de obtencao de plantas geneticamente modificadas de cafeeiro para o silenciamento da expressao do gene das ß-galactosidases. Foram obtidos tres calos transgenicos crescendo em meio seletivo com glufosinato de amonio, dentre os quais dois continham o fragmento deste gene. Estes calos encontram-se em fase de formacao de embrioes somaticos e as plantulas resultantes desta regeneracao serao analisadas, a posteriori, para confirmacao da presenca dos transgenes e avaliacao dos efeitos do silenciamento do gene das ß-galactosidases sobre a maturacao de frutos de cafe.

By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line. / Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs. / In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation. / MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition. / Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs. / This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis. / To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays. / Lee, Sharon Ka-wai. / "December 2009." / Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 216-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100

<p>Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.</p><p>The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.</p><p>The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.</p>