This work presents the development of a new highly sensitive and selective chemical assay for poly(ADP-ribose) which is routinely useful for the determination of polymer levels in vivo. This method was used to carefully measure poly(ADP-ribose) levels in normal and in DNA-damaged cells. The results of these studies strongly suggest that synthesis of poly(ADP-ribose) is involved in some aspect of DNA repair. A review of the literature is presented in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in aspects of transcription, in DNA syn thesis, and in DNA repair largely based on evidence from in vitro studies. It is apparent that current methodology has not allowed the routine quantification of poly(ADP-ribose) in vivo, hence the lack of i^n vivo data concerning the function(s) of the polymer. The body of this work presents the development of two chemical methods for the quantification of poly(ADP-ribose) and the application of one of these methods to the measurement of polymer levels in normal and DNA-damaged cells. Preliminary studies are presented on the utilization of combined gas chromatography/mass spectroscopy for the selective quantification of nucleoside derivatives. A second method makes use of the unique chemistry of the polymer for quantification. The polymer was selectively adsorbed to dihydroxyboryl-sepharose which allowed the removal of most RNA, DNA, and protein from the samples. The polymer was hydrolyzed to the unique nucleoside 2'—^-l*'-ribosyladenosine by digestion with venom phosphodiesterase and bacterial alkaline phosphatase. The 1-N^-etheno derivative of ribosyladenosine was formed by reaction with chloroacetaldehyde and this derivative was seperated from other fluorescent species by reversed phase high pressure liquid chromatography.

DNA-encoded peptide libraries are the basis for in vitro selection methods that use various biological systems (phage display; yeast display; mRNA display). Despite the great success of these selection methods, their obvious disadvantage is the limited number of building blocks, which consist of only twenty proteinogenic amino acids. The involvement of other non- proteinogenic amino acids and other building blocks could significantly expand the range of possible applications of these selection methods. For example, the introduction of chemical modifications in amino acid side chains in such libraries would allow the effective study of post-translational modifications (phosphorylation, acylation, glycosylation, methylation, etc.) in living organisms. The aim of this work was to develop a method for preparation of a fully synthetic DNA encoded library of peptides. The basic steps for the preparation were the chemical synthesis of the peptide and associated enzymatic synthesis of encoding DNA. Compatibility of chemical reactions with DNA is essential for the synthesis of DNA-encoded peptide libraries. Because the final acidic deprotection of the side chains in the peptide is not compatible with DNA, two approaches have been tested to overcome this problem. The first was an attempt to develop finer...

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The use of conventional culture-based approach results in vast majority of microbes (90 - 99%) unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and endonuclease), which are central in genetic engineering. They are usually grouped into four broad classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the extracted metagenomic DNA was used for sequencing and another for fosmid library construction. Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector. A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly, the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the developed DNA-manipulating enzymes and study the practical application and chart way for commercialization. Moreover, the constructed fosmid library could also be screened for potentially novel thermo-stable biomolecules of industrial and therapeutic importance.

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No / The pharmacology of bimatoprost, a synthetic prostaglandin-amide, was examined in prostaglandin F2¿ (PGF2¿)-sensitive preparations. Bimatoprost potently contracted the rabbit isolated uterus (pEC50=7.92±0.16). In contrast, bimatoprost exhibited weak excitatory activity in human myometrium from pregnant and nonpregnant donors, mouse uterus, rat uterus, and endothelium-intact rabbit jugular veins, and did not stimulate DNA synthesis in mouse fibroblasts. The possibility that the effects of bimatoprost may reflect partial agonism at prostanoid FP receptors was examined and the contractile effects of full agonists, 17-phenyl PGF2¿ (FP) and U-46619 (TP, a control), were determined in the absence and presence of 1 ¿M bimatoprost on the mouse uterus. Analyses of the agonist¿agonist functional studies showed no antagonism, indicating that bimatoprost is not a partial agonist. Bioassay metabolism studies of bimatoprost and latanoprost (FP receptor agonist prodrug) in the rabbit uterus were conducted using recipient mouse uterus. Results indicated that the potent responses to bimatoprost in the rabbit uterus are produced by the intact molecule and not by its putative free acid metabolite, 17-phenyl PGF2¿. Some hydrolysis of latanoprost to latanoprost free acid appears to have occurred in the rabbit uterus, according to biological detection. The pharmacology of bimatoprost could not be explained by its interaction with known prostanoid FP receptors and was independent of species-, tissue-, or preparation-related factors. The potent contractile effects of bimatoprost in the rabbit uterus provide further pharmacological evidence for the presence of a novel receptor population that preferentially recognises bimatoprost.

Lau Lai Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-141). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Research initiative and significance --- p.1 / Chapter 1.2 --- Terminal differentiation --- p.4 / Chapter 1.3 --- Controversial terminal differentiation in cardiomyocytes --- p.5 / Chapter 1.4 --- Molecular switch from hyperplasia to hypertrophy in neonatal myocardial development --- p.7 / Chapter 1.5 --- Interferons --- p.8 / Chapter 1.6 --- Functions induced by interferons --- p.9 / Chapter 1.7 --- Interferons in cardiomyocytes --- p.12 / Chapter 1.8 --- Interferon-inducible transmembrane gene family --- p.13 / Chapter 1.9 --- Our hypothesis and objective --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Sequence analysis --- p.18 / Chapter 2.2 --- Cell culture --- p.18 / Chapter 2.3 --- Induction of differentiation of H9C2 cells --- p.19 / Chapter 2.4 --- In vitro induction of IFITMs by interferon treatments --- p.19 / Chapter 2.5 --- RNA isolation --- p.20 / Chapter 2.5.1 --- Experimental animals and sampling --- p.20 / Chapter 2.5.2 --- Total RNA Isolation --- p.20 / Chapter 2.5.3 --- RNA Quantification and Quality Check --- p.21 / Chapter 2.5.4 --- Purification by Qiagen-RNeasy Column and DNase I Digestion --- p.21 / Chapter 2.6 --- First-strand cDNA synthesis --- p.22 / Chapter 2.7 --- Quantitative real-time polymerase chain reaction --- p.22 / Chapter 2.8 --- Cloning protocol --- p.25 / Chapter 2.8.1 --- "Construction of pEGFP-IFITMl, pEGFP-IFITM2 and pEGFP-IFITM3 fusion proteins" --- p.25 / Chapter 2.8.1.1 --- Amplification of DNA fragments --- p.25 / Chapter 2.8.1.2 --- Purification of PCR product --- p.26 / Chapter 2.8.1.3 --- Restriction endonuclease digestion --- p.26 / Chapter 2.8.1.4 --- Insert/vector ligation --- p.27 / Chapter 2.8.1.5 --- Preparation of chemically competent bacterial cells --- p.27 / Chapter 2.8.1.6 --- Transformation of ligation product into chemically competent bacterial cells DH5a --- p.28 / Chapter 2.8.1.7 --- Recombinant clone screening by PCR --- p.29 / Chapter 2.8.1.8 --- Small-scale preparation of recombinant plasmid DNA --- p.29 / Chapter 2.8.1.9 --- Dideoxy DNA sequencing --- p.30 / Chapter 2.8.1.10 --- Large-scale preparation of recombinant plasmid DNA --- p.30 / Chapter 2.8.2 --- "Construction of IFITMl-pcDNA4, IFITM2-pcDNA4 and IFITM3- pcDNA4 constructs" --- p.33 / Chapter 2.8.2.1 --- Amplification of DNA fragments --- p.33 / Chapter 2.8.2.2 --- Insert/vector ligation --- p.33 / Chapter 2.8.2.3 --- Transformation of ligation product into one shot® TOP1 OF´ة chemically competent E. coli cells --- p.34 / Chapter 2.9 --- Transient transfection --- p.36 / Chapter 2.10 --- Subcellular fractionation --- p.37 / Chapter 2.11 --- Isolation of total protein cell lysate --- p.38 / Chapter 2.12 --- Protein concentration determination --- p.38 / Chapter 2.13 --- Protein gel electrophoresis and western blotting --- p.39 / Chapter 2.13.1 --- Preparation of SDS-polyacrylamide gel --- p.39 / Chapter 2.13.2 --- Preparation of protein samples --- p.39 / Chapter 2.13.3 --- SDS-polyacrylamide gel electrophoresis --- p.40 / Chapter 2.13.4 --- Protein transfer to nylon membrane --- p.40 / Chapter 2.13.5 --- Antibodies and detection --- p.40 / Chapter 2.13.6 --- Stripping membrane --- p.41 / Chapter 2.14 --- Bromodeoxyuridine proliferation assay --- p.42 / Chapter 2.14.1 --- Bromodeoxyuridine labeling and detection --- p.42 / Chapter 2.14.2 --- Cell number determination --- p.42 / Chapter 2.15 --- Fluorescence microscopy --- p.43 / Chapter 2.16 --- Confocal microscopy --- p.43 / Chapter 2.17 --- Statistical analysis --- p.44 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequence analysis --- p.45 / Chapter 3.1.1 --- Primary structure analysis --- p.45 / Chapter 3.1.2 --- Transmembrane he lice prediction --- p.46 / Chapter 3.1.3 --- Conserved domain prediction --- p.51 / Chapter 3.1.4 --- Sequence alignments across different species --- p.52 / Chapter 3.2 --- Differential expression during rat myocardial development --- p.53 / Chapter 3.3 --- Altered mRNA levels during differentiation of H9C2 cells --- p.55 / Chapter 3.4 --- "Cloning of IFITMl, IFITM2 and IFITM3" --- p.60 / Chapter 3.5 --- Subcellular localization --- p.61 / Chapter 3.5.1 --- Fluorescence microscopy --- p.61 / Chapter 3.5.2 --- Subcellular fractionation --- p.70 / Chapter 3.6 --- "In vitro induction by interferons-α, β and γ" --- p.72 / Chapter 3.7 --- "DNA synthesis after in vitro induction of interferons-α, β and γ" --- p.79 / Chapter 3.8 --- "Proliferating cell nuclear antigen expression after in vitro induction of interferons-α, β and γ" --- p.87 / Chapter 3.9 --- "DNA synthesis after overexpression of IFITM1, IFITM2 and IFITM3" --- p.93 / Chapter 3.10 --- "Proliferating cell nuclear antigen expression after overexpression of IFITM1, IFITM2 and IFITM3" --- p.95 / Chapter 3.11 --- "β-catenin and cyclin D1 expression after in vitro induction of interferons-α, β and γ" --- p.97 / Chapter 3.12 --- "β-catenin and cyclin D1 expression after overexpression of IFITMl, IFITM2 and IFITM3" --- p.101 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- "Upregulation of IlFITMl, IFITM2 and IFITM3 during myocardial development" --- p.103 / Chapter 4.2 --- "Subcellular localization of IFITMl, IFITM2 and IFITM3" --- p.105 / Chapter 4.3 --- "Induction by interferons-α, β and γ" --- p.107 / Chapter 4.4 --- Inhibition of DNA synthesis by interferons-α and β and IFITM1 --- p.109 / Chapter 4.5 --- Involvement of IFITM family in canonical Wnt pathway --- p.112 / Chapter 4.6 --- Other possible pathways involved --- p.117 / Chapter CHAPTER 5 --- FUTURE PROSPECTS / Chapter 5.1 --- Production of antibodies --- p.118 / Chapter 5.2 --- Silencing or knockout approach --- p.118 / Chapter 5.3 --- Target genes of Wnt/β-catenin signaling --- p.119 / Chapter 5.4 --- Other signaling pathways involved --- p.119 / Chapter 5.5 --- Use of primary cardiomyocytes --- p.120 / APPENDIX --- p.121 / REFERENCES --- p.124

The evolutionary pressure to prevent re-replication by inactivating licensing during S phase leaves higher-eukaryotes with large genomes, such as human cells, vulnerable to replication stresses. Origins licensed in G1 must be sufficient to complete replication as new origins cannot be licensed in response to irreversible replication fork stalling. Interdisciplinary approaches between cellular biology and biophysics predict that replication of the genome is routinely incomplete in G2, even in the absence of external stressors. The frequency of converging replication forks that never terminate due to irreversible stalling (double fork stall), which result in a segment of unreplicated DNA, was modelled using high quality origin-mapping data in HeLa and IMR-90 cells. From this, hypotheses were generated that related an increase in unreplicated segments of DNA to reduced functional origin number. Presented in this thesis is the confirmation of this relation by quantifying chromosome mis-segregation and DNA damage responses when origin number was reduced using RNAi against licensing factors. The number of ultrafine anaphase bridges and 53BP1 nuclear bodies are in remarkable concordance with the theoretical predictions for the number of double fork stalls, indicating that cells are able to tolerate under-replication through such post-replicative cellular responses. 53BP1 preferentially binds to chromatin associated with large replicons, and functions synergistically with dormant origins to protect the stability of the genome. Additional candidates, inspired by common fragile site research, have also been characterised as responders to spontaneous under-replication, and include FANCD2 and MiDAS, which function in early mitosis to facilitate completion of replication before cells enter anaphase. In conclusion, a series of mechanisms that sequentially function throughout the cell cycle protects the stability of the human genome against inevitable spontaneous under-replication brought about by its large size.

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