No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 290-308).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 239-251).

Eicosapentaenoic acid (EPA, C20:5, n-3) is a long chain crucial unsaturated fatty acid, essential for the regulation of critical biological functions in humans. Its benefits include the therapeutic treatment of cardiovascular disease, schizophrenia and Alzheimer's disease. The fungus Pythium irregulare (ATCC 10951) has great potential as a natural EPA producer. In this study, the electroporation conditions for P. irregulare were determined. The auxotrophic selectable genes ura, trp and his were respectively cloned into the plasmid pESC to construct shuttle vectors. Electroporation with 2.0kV and a 0.2cm cuvette was applied as the most effective condition for heterogeneous genes transformation. The yield and content of EPA and other components of total fatty acids (TFA) were further determined by the FAME approach with GC, as well as the analysis of biomass. The EPA content in P. irregulare with heterologous pESC-TRP vector reached 16.68 mg/g if cultured in auxotrophic medium, which showed a 52.33% increase compared to the wild-type P. irregulare. The maximum of EPA yield was 98.52 mg/L from P. irregulare containing the pESC-URA plasmid, a 32.28% increase over the wild-type. However, the maximum cell dried weight of these two organisms were respectively 6.13g/L and 5.3g/L, significantly less than the 6.80g/L of the wild-type. Not only was a feasible approach detected to electro-transform and increase the EPA yield of P. irregulare, this study also inferred that Ï -6 route was mainly involved in the EPA biosynthesis in this organism. An antisense RNA (asRNA) thermodynamic model was developed to design new asRNA constructs capable of fine-tuning gene expression knockdown. The asRNA technology is now identified as an effective and specific method for regulating microbial gene expression at the posttranscriptional level. This is done by targeting mRNA molecules. Although the study of regulation by small RNAs is advanced in eukaryotes, the regulation of expression through artificially introducing antisense oligodeoxynucleotides into host is still being developed in prokaryotes. To study the thermodynamics of asRNA and mRNA binding, (i) the fluorescence protein genes GFP and mCherry were separately cloned into the common pUC19 vector and (ii) antisense GFP and antisense mCherry DNA fragments were randomly amplified and inserted into the constructed plasmid under the control of an additional plac promoter and terminator. The expression level of fluorescence reporter proteins was determined by plate reader in this combinatorial study. A thermodynamic model to describe the relationship between asRNA binding and observed expression level was created. The study indicates two factors that minimum binding energy of the asRNA-mRNA complex and the percentage of asRNA binding mRNA were crucial for regulating the expression level. The correlation relationship between gene expression level and binding percentage multiplied by the minimum binding energy was found to show a good correlation between the thermodynamic parameters and the observed level of gene expression. The model has the potential to predict the sequence of asRNA and the approach will ultimately be applied to cyanobacteria to increase lipids production. Here, the long-term approach is to build metabolic switches from asRNA that can turn "on/off" various cellular programs and metabolic pathways at will in a fine-tuned manner. This will allow engineers to control metabolic activity in response to reactor conditions. / Master of Science

Progress in the biological sciences relies on the availability of appropriate tools and strategies; in the areas of understanding and constructing multicellular systems, better methods of gene expression control are needed. Here, we work towards two approaches to controlling gene expression for applications related to multicellular coordination and pattern formation. First, we characterize the use of 2A self-cleaving peptides for polycistronic expression in Dictyostelium discoideum, a powerful model system for investigating the onset of coordinated behavior in a cell population. We also make novel observations about various factors that affect gene expression levels, which would inform future decisions on the use of 2A peptides in this system. Second, we examine the use of antisense RNA in mammalian cells for gene repression from an input that simultaneously activates other genes. We show that in transient overexpression systems, antisense RNA produced in trans can be a more effective repressor than convergent promoters and other well-established RNA-based methods for gene repression, and we explore ways in which this system could be improved for stable gene circuits. Overall, our results advance our understanding of available tools for gene expression control and the conditions under which they are appropriate to use.

Yau, Pak Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-167). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / List of abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Table of content --- p.xii / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1. --- Introduction / Chapter 1.1.1. --- Tumor hypoxia --- p.2 / Chapter 1.1.2. --- Non-coding RNA --- p.6 / Chapter 1.1.3. --- Long non-coding RNAs: regulation and related diseases --- p.7 / Chapter 1.1.3.1. --- aHIF and cancer --- p.11 / Chapter 1.1.4. --- Objective --- p.11 / Chapter Chapter Two: --- Regulation of HIF-lα by aHIF --- p.13 / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- aHIF: a natural antisense long non-coding RNAs --- p.14 / Chapter 2.1.2. --- The relationship between aHIF and HIF-lα --- p.15 / Chapter 2.1.3. --- HIF-lα regulation --- p.19 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Cell culture --- p.22 / Chapter 2.2.2. --- Western blot analysis --- p.22 / Chapter 2.2.3. --- RNA isolation and reverse transcription --- p.23 / Chapter 2.2.4. --- Quantitative Real-time PCR --- p.23 / Chapter 2.2.5. --- Plasmids construction --- p.24 / Chapter 2.2.5.1. --- aHIF over-expression clone --- p.24 / Chapter 2.2.5.2. --- Luciferase reporter with HIF-lα 3'UTR --- p.25 / Chapter 2.2.5.3. --- HIF-lα and PTB over-expression vector --- p.25 / Chapter 2.2.5.4. --- PTB knock-down vector --- p.30 / Chapter 2.2.6. --- Stable Clone --- p.30 / Chapter 2.2.7. --- Transfection --- p.31 / Chapter 2.2.8. --- Luciferase reporter assay --- p.31 / Chapter 2.2.9. --- Statistical analysis --- p.32 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Effect of aHIF (FL) on HIF-lα expression --- p.33 / Chapter 2.3.2. --- Effect of aHIF (FL) on HIF-lα 3，UTR --- p.33 / Chapter 2.3.3. --- Effects of aHIF (OL) and aHIF (NOL) on HIF-lα level --- p.37 / Chapter 2.3.4. --- Effects of aHIF (NOL) and aHIF (OL) on HIF-lα 3，UTR --- p.39 / Chapter 2.3.5. --- Effect of aHIF (FL) on HIF-lα 3' UTR in PTBi cells --- p.41 / Chapter 2.3.6. --- Effect of aHIF (NOL) on HIF-lα 3，UTR in PTBi cells --- p.43 / Chapter 2.3.7. --- Effect of aHIF (OL) on HIF-lα 3' UTR in PTBi cells --- p.45 / Chapter 2.4 --- Discussion / Chapter 2.4.1. --- aHIF regulates HIF-la through HIF-la 3' UTR (FL) --- p.47 / Chapter 2.4.2. --- Factors involved in aHIF- HIF-lα interaction --- p.53 / Chapter Chapter Three: --- aHIF regulates drug sensitivity through BNIP3 --- p.58 / Chapter 3.1 --- Introduction / Chapter 3.1.1. --- aHIF and drug sensitivity --- p.59 / Chapter 3.1.2. --- BNIP3: its regulation and functions --- p.61 / Chapter 3.1.3. --- Taxol and its action mechanism --- p.67 / Chapter 3.1.4. --- Objective --- p.69 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Cell culture --- p.70 / Chapter 3.2.2. --- Cell viability assay --- p.70 / Chapter 3.2.3. --- Western blot analysis --- p.70 / Chapter 3.2.4. --- Plasmid construction --- p.71 / Chapter 3.2.5. --- Transfection --- p.71 / Chapter 3.2.6. --- Stable clone formation --- p.71 / Chapter 3.2.7. --- Quantitative real-time PCR --- p.71 / Chapter 3.2.8. --- Annexin V binding assay --- p.72 / Chapter 3.2.9. --- DNA fragmentation assay --- p.72 / Chapter 3.2.10. --- Detection of mitochondrial membrane potential by flow cytometry --- p.73 / Chapter 3.2.11. --- Cytochrome c and AIF translocation assay --- p.73 / Chapter 3.2.12. --- Statistical analysis --- p.74 / Chapter 3.3 --- Results / Chapter 3.3.1. --- Effect of aHIF on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.2. --- Effect of HIF-lαi on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.3. --- Effect of aHIF on Taxol-induced apoptosis --- p.78 / Chapter 3.3.4. --- HIF-1α regulation of BNIP3 expression --- p.78 / Chapter 3.3.5. --- Effect of aHIF on BNIP3 expression --- p.81 / Chapter 3.3.6. --- BNIP3 expression in BNIP3i stable transfectant --- p.81 / Chapter 3.3.7. --- The response of BNIP3i cells towards Taxol and vincrisinte --- p.84 / Chapter 3.3.8. --- Effect of BNIP3 on Taxol and vincristine sensitivity in BNIP3i cells --- p.84 / Chapter 3.3.9. --- Taxol- or vincristine- induced apoptosis in BNIP3i cells --- p.87 / Chapter 3.3.10. --- "Effects of aHIF, HIF-lα and BNIP3 on Taxol-induced apoptosis in HepG2 cells" --- p.89 / Chapter 3.3.11. --- Caspases activation in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.12. --- Mitochondrial membrane depolarization in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.13. --- AIF and cytochrome c expressions in BNIP3i cells --- p.92 / Chapter 3.3.14. --- Effect of aHIF on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.15. --- Effect of HIF-lα on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.16. --- BNIP3i cells became more sensitivity to a number of drugs --- p.99 / Chapter 3.3.17. --- BNIP3i became more resistance to some drugs --- p.99 / Chapter 3.4 --- Discussion / Chapter 3.4.1. --- aHIF affected Taxol sensitivity through BNIP3 --- p.102 / Chapter 3.4.2. --- Mechanism of BNIP3 regulated Taxol or vincristine induced apoptosis --- p.106 / Chapter 3.4.3. --- Possible roles of BNIP3 in response to other therapeutics --- p.110 / Chapter Chapter Four: --- aHIF regulation of tumorigenesis --- p.116 / Chapter 4.1 --- Introduction / Chapter 4.1.1. --- aHIF in cancer biology --- p.117 / Chapter 4.1.2. --- Ras proteins --- p.118 / Chapter 4.1.3. --- K-Ras and cancers --- p.121 / Chapter 4.1.4. --- Regulation of Ras --- p.122 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1. --- Cell culture --- p.124 / Chapter 4.2.2. --- Western blot analysis --- p.124 / Chapter 4.2.3. --- Plasmids construction --- p.124 / Chapter 4.2.4. --- Transfection --- p.124 / Chapter 4.2.5. --- Cell growth assay --- p.124 / Chapter 4.2.6. --- Soft agar assay --- p.125 / Chapter 4.2.6. --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1. --- Effect of aHIF and HIF-lα on cell proliferation --- p.127 / Chapter 4.3.2. --- Effect of aHIF and HIF-lα on anchorage-independent growth --- p.127 / Chapter 4.3.3. --- Effect of aHIF and HIF -lα on K-Ras expression --- p.130 / Chapter 4.3.4. --- Effect of FTS on cell transfected with aHIF or HIF-lα --- p.130 / Chapter 4.4 --- Discussion / Chapter 4.4.1. --- Role of aHIF in tumorigenesis --- p.133 / Chapter 4.4.2. --- Proposed pathways of aHIF-regulated tumorigenesis --- p.136 / Chapter Chapter Five: --- General discussion and conclusion --- p.140 / Chapter 5.1 --- General discussion --- p.141 / Chapter 5.2 --- Conclusion --- p.146 / Chapter 5.3 --- Future perspectives --- p.147 / Chapter 5.3.1 --- Role ofPTB in aHIF-HIF-lα interaction --- p.147 / Chapter 5.3.2 --- Effect of aHIF (OL) on HIF-lα mRNA 3' UTR --- p.147 / Chapter 5.3.3 --- Effect ofaHIF on AIF --- p.148 / Chapter 5.3.4 --- Confirmation of the involvement of K-Ras --- p.148 / Chapter Chapter Six --- References --- p.150 / Chapter 6.1 --- References --- p.151

<p>Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. </p><p>In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using <i>in vivo</i> reporter gene fusion expression and different <i>in vitro </i>methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.</p><p>Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.</p><p>Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.</p>

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays. Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn. Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.