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by Agnes, Lo Shuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves [27-33] (2nd gp.)). / Chapter PART 1: --- Human Heart cDNA Library Sequencing / Chapter A) --- Introduction of human heart cDNA library sequencing / Chapter A.1 --- Human genome project / Chapter A.2 --- The aim of human genome project / Chapter A.3 --- Automatic sequencing / Chapter A.4 --- Cycle sequencing reaction / Chapter A.5 --- Human heart cDNA library sequencing project / Chapter B) --- Methods and materials / Chapter (I) --- Preparation of plating bacterial-Y1090 / Chapter (II) --- Plating the bacteriophage with blue-white visual selection / Chapter (III) --- Amplification of bacteriophage cDNA clones by PCR / Chapter (IV) --- Purification and quantitation of PCR products / Chapter (V) --- Cycle DNA sequencing of PCR products / Chapter (VI) --- Casting the sequencing gel / Chapter (VII) --- Sequencing by Pharmacia LKB A.L.F. DNA Sequencer / Chapter (VIII) --- Editing and saving the DNA sequence / Chapter (IX) --- Sending the DNA sequence to Genbank by E-mail / Chapter (X) --- Usage of the Genbank database / Chapter C) --- Results / Chapter D) --- Discussions / Chapter D.1 --- Application of human genomic project / Chapter D.2 --- Interpretation of the sequencing results / Chapter D.3 --- Quality of cDNA libraries and representation of mRNA population / Chapter D.4 --- "Gene expression profile in three different organs-heart, brain and liver" / Chapter D.5 --- Population study of the cDNA library / Chapter D.6 --- Isolation of a large number of novel genes by substraction cDNA library / Chapter D.7 --- Screening method to find out the complete coding sequence of interesting genes / Chapter D.8 --- Technical problems encountered and managed / Chapter PART 2: --- Identification of human cytosolic malate dehydrogenase by large scale human heart cDNA library sequencing / Chapter CHAPTER 1: --- Introduction of malate dehydrogenase / Chapter 1.1 --- Malate dehydrogenase--Kreb's cycle enzyme / Chapter 1.2 --- Two stereospecific forms of dehydrogenase / Chapter 1.3 --- NAD-binding domain / Chapter 1.4 --- The active site / Chapter 1.5 --- Comparison of surface properties between cMDH and mMDH / Chapter 1.6 --- N-terminal region and mitochondrial import / Chapter 1.7 --- Subunit-subunit interactions / Chapter 1.8 --- Physiological importance of malate dehydrogenase / Chapter 1.9 --- Secondary structure-total 11 β-strands and 9 α-helixes / Chapter 1.10 --- Objectives of the thesis / Chapter CHAPTER 2: --- Cloning and sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1 --- Cloning of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1.1 --- Methods and materials / Chapter 2.1.1.1 --- Cloning full length of hcMDH into expression vector pAED4 / Chapter 2.1.1.2 --- Preparation of competent cell-JM109 for transformation / Chapter 2.1.1.3 --- Minipreparation of plasmid DNA / Chapter 2.1.1.4 --- Midi-preparation of bacteriophage λDNA by QIAGEN´ёØ / Chapter 2.1.1.5 --- Titration of bacteriophage λ of human adult heart cDNA library / Chapter 2.1.1.6 --- Preparation of soft-agarose lysates / Chapter 2.1.1.7 --- Elution of DNA from agarose gel by GENECLEAN´ёØ / Chapter 2.1.2 --- Results / Chapter 2.1.3 --- Discussions / Chapter 2.2 --- Sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.2.1 --- Methods and materials: Autoread sequencing / Chapter (I) --- Annealing of primer to double-stranded template / Chapter (II) --- Sequencing / Chapter 2.2.2 --- Results and discussions / Chapter 2.3 --- Amino acids and protein structure analysis of cMDH / Chapter CHAPTER 3 : --- "Protein expression, partial purification and folding experiments of human cytosolic malate dehydrogenase (hcMDH)" / Chapter 3.1 --- Protein expression of hcMDH in E. coli / Chapter 3.1.1 --- Methods and materials / Chapter 3.1.1.1 --- Protein expression induced by IPTG / Chapter 3.1.1.2 --- Isoelectric focusing (IEF)-two dimensional gel electrophoresis / Chapter (I) --- First dimensional electrofocusing / Chapter (II) --- The second dimension SDS-PAGE electrophoresis / Chapter (III) --- Sample preparation / Chapter 3.1.2 --- Results / Chapter 3.1.3 --- Discussions / Chapter 3.1.3.1 --- The properties of expressed protein of hcMDH / Chapter 3.1.3.2 --- T7 expression system / Chapter 3.1.3.3 --- Strong φ 10 promoter / Chapter 3.1.3.4 --- E.coli BL21 host cell / Chapter 3.2 --- Partial purification and folding experiments of hcMDH / Chapter 3.2.1 --- Methods and materials / Chapter 3.2.1.1 --- Partial purification of hcMDH expressed protein / Chapter (I) --- Preparation of supernatant from E.coli crude extract / Chapter (II) --- Ion-exchange column chromatography / Chapter (III) --- Affinity chromatography / Chapter (IV) --- Gel filtration on a Sepharose CL-6B column / Chapter 3.2.1.2 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 3.2.1.3 --- Staining the protein gel by the Coomassie Blue R-250 method / Chapter 3.2.1.4 --- Staining the protein gel by the Silver staining Method / Chapter 3.2.1.5 --- Quantitation of protein by the Bradford Method / Chapter 3.2.1.6 --- Native gel electrophoresis / Chapter 3.2.1.7 --- Malate dehydrogenase MDH enzyme staining method / Chapter 3.2.1.8 --- Malate dehydrogenase MDH enzyme assay / Chapter 3.2.1.9 --- Fast protein liquid chromatography (FPLC) / Chapter 3.2.1.10 --- Protein folding experiment / Chapter 3.2.1.11 --- Eukaryotic expression of hcMDH / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Partial purification by chromatography / Chapter 3.2.2.2 --- Native gel / Chapter 3.2.2.3 --- FPLC / Chapter 3.2.2.4 --- To aid folding of protein by adding NADH / Chapter 3.2.2.5 --- Eukaryotic expression / Chapter 3.2.3 --- Discussions / Chapter 3.2.3.1 --- Purification of malate dehydrogenase MDH / Chapter 3.2.3.2 --- "Methods for visualizing dehydrogenase enzymes, e.g. malate dehydrogenase" / Chapter 3.2.3.3 --- The presence of unfold hcMDH protein in bacteria / Chapter 3.2.3.4 --- Folding of protein by heat shock protein GroE / Chapter 3.2.3.5 --- Eukaryotic expression / Chapter CHAPTER 4: --- Master screening of single base change by PCR-SSCP (Single Strand Conformational Polymorphism) / Chapter 4.1 --- Theory of SSCP / Chapter 4.2 --- Methods and materials / Chapter 4.3 --- Results / Chapter 4.4 --- Discussions / Chapter 4.4.1 --- The procedure of SSCP / Chapter 4.4.2 --- An alternative quick detection method for polymorphism of hcMDH at position 565--by automatic sequencing / Chapter 4.4.3 --- Other detection methods-- RNA-PCR and ddF / Chapter 4.4.4 --- Parameters affecting sensitivity of SSCP / Chapter 4.4.5 --- Application of SSCP / Chapter CHAPTER 5: --- Southern hybridization and In situ hybridization / Chapter 5.1 --- Southern blot analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 5.1.1 --- Methods and materials / Chapter (I) --- Transfer genomic DNA to Nylon membrane / Chapter (II) --- Synthesis of radiolabelling cDNA probe / Chapter (III) --- Pre-hybridization and hybridization reaction / Chapter 5.1.2 --- Results / Chapter 5.1.3 --- Discussions / Chapter 5.2 --- In situ hybridization / Chapter 5.2.1 --- Methods and materials / Chapter (I) --- Preparation of Dig labelling probe by random primed labelling / Chapter (II) --- Estimating the yield of Dig-labelled nucleic acids / Chapter (III) --- Denaturation and hybridization of the hcMDH probe with animal tissues / Chapter (IV) --- Color development of the tissue / Chapter 5.2.2 --- Results / Chapter 5.2.3 --- Discussions / Chapter 5.2.3.1 --- Cellular distribution of hcMDH / Chapter 5.2.3.2 --- The principle of in situ hybridization / Chapter 5.2.3.3 --- Specimen preparation / Chapter 5.2.3.4 --- Hybridization conditions / Chapter 5.2.3.5 --- "Ontogeny of MDH in rabbit fetal brain, heart and lung" / Appendixes: / "Appendix I: 531 random cDNA clones from clone no. J950 to K951 in human heart cDNA library sequencing project. The name of clones, accession number, the length of the partial sequence and percentage of match are listed" / Appendix II: The new accession no. of Novel clones in Genbank / "Appendix III: The enzymatic reaction, molecular weigth, specific activity and Michaelis constants of different sources of malate dehydrogenase" / Appendix IV: The full sequence of nucleic acids and amino acids of human cytosolic malate dehydrogenase hcMDH. Accession no. of hcMDH is U20352 in Genbank / Appendix V: Nucleotide sequences of the mouse cMDH gene / Appendix VI: Nucleotide sequences of the mouse mMDH gene / Appendix VII: Structural organization of the mouse cytosolic malate dehydrogenase and its comparison with that of the mouse mitochondrial malate dehydrogenase gene

The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures. Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called 'Interferon Stimulatory DNA' or 'ISD') remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA. Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA- binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins. We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA.

Univ., Diss.--Münster, 2004.

Thesis (M.Sc. (Biochemistry)) -- Mahidol University, 1978. / Supported by the University Development Commission and National Research Council.

Thyroid nodules represent the most frequent endocrine lession in our population and it is neccessary to differentiate malignant lessions from them. The aim of the study was to validate determination of selected angiogenic, proliferative, and appptotic markers in cytosol tissue extracts. We analysed 166 tissue samples (85 goitres, benign adenomas and 10 malignat tumours in which VEGF, bFGF, Endostatin, Thymidinkinase ans TPS were determined. Main limitation of cytosolic analysis is tissue sample volume, that must be about 1cm3 and interindividual variability caused by tissue sample heterogeneity. Best way is to compare normal with pathological tissue samples from one patient. We fund significant differences amog histological groups in VEGF, bFGF, Endostatin and maily Tymidinkinase and TPS. These differences are not sufficiently huge to distinguish goitres and benign lessions . We also did not find any correlation between cytosolic markers and iminuhistochemistry markers . Cytosol analysis is not able to measure local expression and its differences in anylysed tissue, but it is able to quantitatively determine mean levels of selected markers.

In light of an urgent need for improved antimicrobial diagnostics and therapeutics, understanding bacterial behaviour, and bacterial responses to treatments in particular, is one of the key objectives of modern medical research. While the molecular mode of action of antibiotics is usually well known, their effect on the cell at a "systems" level (on the regulatory networks, metabolism, etc.) is only beginning to be quantitatively understood. We address some of these response phenotypes in Escherichia coli testing different antibiotic classes and growth conditions. We study the short (< 15 s) time-scale fluctuation dynamics of fluorescently-tagged chromosomal loci and cytosolic aggregates, which report for the state of locus ”compaction” and the levels of macromolecular crowding of the cytosol, respectively. We improve the precision of those measurements developing a novel data treatment procedure and discover that sub-lethal doses of ciprofloxacin, rifampicin, and vancomycin as well as hyperosmotic shock conditions cause small but consistent changes (unique to each treatment agent) to the physical organisation of chromosomal Ori2 and Ter3 loci and the cytosol. We reveal, among other findings, strong correlations between the effects in different parts of the chromosome and between the chromosome and cytosol. In addition, we complement the marker dynamics work with single-cell level gene expression measurements during sub-lethal translation inhibition. Specifically, we compare responses to tetracycline and chloramphenicol from constitutive and ribosomal promoters in Ori3 and Ter3 chromosomal positions over long (7 h) treatment times in exponentially growing bacteria. We reveal, for the first time, the kinetics of cellular resource allocation and provide novel insights on globally regulated transcription, relevant to the three-component proteome partitioning model, gene-length dependent effects of the processivity of translation, and ”reversibility” of ribosome-binding antibiotics. In addition, we discover a strong correlation between the timing of responses from promoters in the Ori3 and Ter3 positions, and a small but consistent difference in the response magnitude between the two positions.

published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy

Universiẗat, Diss., 2005--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Diss. (sammanfattning) Göteborg : Univ., 2007. / Härtill 3 uppsatser.