Wang, Dakui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 208-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Glutamine synthetase (GS; EC 6.3.1.2; L-glutamate ammonialigase) catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine. Due to its key role in nitrogen metabolism, including nucleotide, amino acid and urea biosynthesis, the enzyme has been ascribed an extraordinarily long evolutionary history. Thus, GS has been used as a molecular clock to establish phylogenetic relationship between different species. Through the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) system, we obtained the complete cDNA of GS from tilapia cDNA liberary. Furthermore, the results of the alignment of tilapia GS sequence with that of other species indicated a close relationship between tilapia GS and other fishes. We also found that there is 79% homology between mammal and tilapia within the open read frame (ORF) of GS. However, sequence analysis by computer software revealed the fact that the size (0.5 kb) of GS 3¡¦untranslated region (3¡¦-UTR) of tilapia GS is different from that of mammals. Moreover, there is the complete distinct sequence of the 3¡¦-UTR of tilapia GS from that of mammals. The 3'-UTR of many eukaryotic mRNAs has been implicated in the control of mRNA stability, processing, polyadenylation, and translational regulation. Accordingly, to comprehend the role of 3¡¦-UTR in GS phylogenesis, we examine whether the 3'-UTR of tilapia GS is involved in the regulation of GS expression in mammals. We first generated the construct using pEGFP-N2 carrying the ORF (1.1kb) of tilapia GS gene (ORF-GFP) or the full length (1.6kb) of tilapia GS gene (Full-GFP). Transient or stable transfection of C6 gliomal cells with ORF-GFP indicated that GS mRNA and protein was expressed. When C6 cells were stably transfected with Full-GFP, the expression of GS mRNA, but not its protein, was found. Adenine/uridine-rich sequence elements (AREs) of the 3¡¦-UTR have been known to regulate mRNA stability of certain chemokines. Four AREs are also found in the 3¡¦-UTR of tilapia GS. We further generated the constructs with tilapia ORF-GFP and its 3¡¦-UTR containing 1-4 AREs (A1-GFP, A2-GFP, A3-GFP and A4-GFP). Stable transfection of C6 cells with the different constructs indicated that tilapia GS mRNA is normally transcripted, while there was no expression of GS proteins in stable transfectants. The findings suggest tilapia GS protein expression in mammals by its 3¡¦-UTR and unidentified evolutionary role of the 3¡¦-UTR region of GS.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 198-225). Online version available via ProQuest Digital Dissertations.

Thesis (M.S.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences (Bioinformatics and Proteomics-Genomics)." Title from title page of PDF document. Bibliography: p. 52-57.

Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.

Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.

The microtubule-associated protein tau forms pathological neuronal filaments in Alzheimer's disease (AD) and other neurodegenerative disorders, known collectively as tauopathies. Previous studies in transgenic mouse models of AD suggest that reducing tau expression may be safe and beneficial for the prevention or treatment of AD and possibly other tauopathies. As a first step toward identifying novel therapeutic strategies to reduce tau levels, the studies presented in this dissertation aim to investigate the role of the human tau 3'-untranslated region (3'-UTR) in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. The exact sequence of these two 3'-UTR isoforms was determined by rapid amplification of cDNA 3'-ends (3'-RACE), and the two 3'-UTR isoforms were cloned into a luciferase reporter vector. Using these reporter constructs, the expression of these isoforms was found to be differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y by luciferase assays and quantitative PCR (qPCR). Through an unbiased screen of tau 3'-UTR deletions and fragments using luciferase reporter constructs, several regions in the long tau 3'-UTR isoform that contain regulatory cis-elements were identified. Additionally, several microRNAs were computationally identified as candidates that might bind the long tau 3'-UTR and thereby differentially control the expression of long versus short tau 3'-UTR isoforms. Screening these candidate microRNAs via luciferase reporter assay identified miR-34a, which was subsequently shown to repress the expression of endogenous tau protein and mRNA in M17D cells using Western blot and qPCR, respectively. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. Taken together, these studies suggest that the expression of the two tau 3'-UTR isoforms is differentially regulated and that this differential regulation is due to the presence of regulatory cis-elements found only in the long tau 3'-UTR isoform, including a binding site for miR-34 family members. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies.

Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease. / text

Non-coding RNAs (ncRNAs) have important biological functions such as regulation of gene expression and disease causality. These ncRNAs exert their functions by interacting with other molecules, such as messenger RNAs (mRNAs). Thus RNA-RNA interaction studies are important for understanding the gene regulation mechanism and for curing ncRNAs related diseases. This thesis contributes to RNA-RNA interaction prediction problem, construction of a comprehensive human micro RNAs (miRNAs)-related database and data mining on high throughput RNA-RNA interaction data. / On RNA-RNA interaction prediction problem, a novel energy model is proposed and a GA based algorithm is developed, namely RIPGA. The experiments results show that the novel energy model outperforms the state of the art model, which is called Turner energy model, RIPGA with novel energy model also outperforms two state of the art programs, which are called inRNAs and RactIP. / On construction of a comprehensive human miRNA-related database, data are collected and cleansed from three state of the art databases related to human miRNAs, which are called miR-TarBase, miRBase and HMDD v2.0. A network is constructed from these data to present the complete relationships of the miRNAs because the relationships are only partial covered by the existing databases. A website and database are setup for data query, visualization and analysis functions to complement the existing databases. / On data mining on high-throughput RNA-RNA interaction data, four characteristics of RNA-RNA interaction are identified from the high-throughput data. We believe these characteristics are potential explanations of the high degree of connectivity of some miRNAs. These characteristics are also important scientific knowledge for future research on RNA-RNA interaction and control for biomedical applications. / 非編碼核糖核酸(ncRNAs)有重要的生物功能，例如：基因表現的調控和疾病的因果關係。這些ncRNAs透過與其他分子的互相作用來發揮作用，例如：信使核糖核酸(mRNAs)。因此，核糖核酸互相作用的研究對理解基因表現的調控和治愈與ncRNAs有關的疾病十分重要。本論文集中解決核糖核酸互相作用的預測問題、建設人類微核糖核酸(miRNAs)綜合數據庫和高通量核糖核酸互相作用數據的數據挖掘。 / 針對核糖核酸互相作用的預測問題，我們提出了一個新的能量模型和開發了一個基於遺傳算法的算法，即RIPGA。實驗數據顯示新的能量模型比最先進的模型﹐即特納能量模型做得更好。而RIPGA亦比最先進的inRNAs和RacIP做得更好。 / 針對建設人類微核糖核酸綜合數據庫，我們收集及潔淨來自三個最先進的數據庫的數據，即miRTarBase，miRBase和HMDDv2.0。我們由這些數據建設了一個網絡來表達完整的miRNAs關係，因為現存的數據庫只覆蓋了部份的關係。我們亦設置了網站和數據庫，並提供數據查詢、可視化及分析的功能，以補足現存的數據庫。 / 針對高通量核糖核酸互相作用數據的數據挖掘，我們從高通量核糖核酸互相作用數據中認出四個核糖核酸互相作用的特點。我們相信這些特點是部份高連通miRNAs的可能的解釋。這些特點對核糖核酸互相作用及控制的未來研究和生物醫學應用亦是重要的科學知識。 / Cheung, Kwan Yau. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 149-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Eukaryotic gene expression is a complex process and can be controlled at the level of transcription, post-transcription or translation and post-translation. In recent years, there is a growing interest in understanding the role of 3'-untranslated region (UTR) in regulating mRNA turnover and translation. The 3'-UTR harbors the poly(A) signal and post-transcriptional regulatory sequences like miRNA and AU-rich elements (AREs). The presence of multiple poly(A) sites often results in multiple transcripts; shorter transcripts correlating with more protein abundance. Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix localized antioxidant enzyme that converts mitochondrial generated superoxide to hydrogen peroxide. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. We hypothesize that the 3'-UTR of MnSOD regulates its mRNA and protein levels as well as activity in response to growth states and environmental stress. Results from a Q-RT-PCR assay showed a preferential accumulation of the shorter MnSOD transcript during quiescence, which correlated with an increase in MnSOD activity. The accumulation of the longer MnSOD transcript during proliferation was associated with a decrease in MnSOD activity. Log transformed expression ratio of the longer to shorter transcript was also higher in proliferating epithelial non-cancerous (mammary: MCF-10A) and cancer cells (mammary: MB-231, SUM 159; oral squamous: SQ20B, FaDu, Cal27; and lung: A549, H292), suggesting that the abundance of the longer transcript is independent of cellular transformation status, instead it is dependent on cellular growth state. Interestingly, the abundance of the longer transcript directly correlated with percent S-phase (R2=0.86). The shorter transcript was enriched in irradiated MB-231 cells. MCF-10A cells exposed to 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of the environmental pollutant polychlorinated biphenyl 3, showed a significant decrease in the abundance of the 4.2 kb transcript due to a faster mRNA turnover, 14 h compared to 20 h in untreated control cells. The decrease in the 4.2 kb transcript levels was associated with a corresponding decrease in MnSOD protein levels and activity, which resulted in a significant inhibition of quiescent cells entry into the proliferative cycle. Deletion and reporter assays showed: (a) a significant decrease in reporter activity in constructs carrying multiple AREs that are present in the 3'-UTR of the longer MnSOD transcript; (b) irradiation increased the reporter activity of the constructs carrying the 3'-UTR sequence of the shorter MnSOD transcript and (c) N-acetyl-cysteine increased the reporter activity of constructs carrying multiple AREs. Because the longer transcript carries AREs, our results identified redox sensitive AREs as novel regulators of MnSOD transcript levels. We conclude that MnSOD 3'-UTR is a novel molecular sensor regulating MnSOD mRNA levels in response to different growth states and environmental stress. A better understanding of the 3'-UTR regulating gene expression could lead to the development of new molecular biology-based redox therapy designed to treat proliferative disorders.