Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 3 uppsatser.

by Au Lai Yin, Cathy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 50-66). / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.x / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Mycobacterial tuberculosis Infections --- p.3 / Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3 / Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4 / Chapter 1. --- Conventional methods --- p.4 / Chapter 2. --- Rapid methods --- p.4 / Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5 / Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6 / Chapter 1. --- Choice of target sequences --- p.6 / Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7 / Chapter 3. --- Studies on pure cultures --- p.9 / Chapter a. --- Detection limit - target DNA --- p.9 / Chapter b. --- Detection limit - Colony forming units --- p.9 / Chapter c. --- Detection limit - Number of cells --- p.10 / Chapter 4. --- Studies on clinical specimens --- p.10 / Chapter 5. --- Problems --- p.12 / Chapter a. --- Availability of target DNA --- p.13 / Chapter (i) --- Cell breakage efficiency --- p.13 / Chapter (ii) --- Target sequence --- p.14 / Chapter b. --- Inhibitory factors for Taq polymerase --- p.14 / Chapter c. --- Contamination --- p.15 / Chapter VIII. --- MATERIALS AND METHODS --- p.16 / Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16 / Chapter 1. --- Reference Strains --- p.16 / Chapter 2. --- Clinical isolates --- p.16 / Chapter B. --- Growth media and culture conditions --- p.17 / Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17 / Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18 / Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19 / Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19 / Chapter 4. --- Southern Blotting --- p.19 / Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20 / Chapter 6. --- Hybridization --- p.21 / Chapter 7. --- Detection --- p.21 / Chapter D. --- Assessment of number of organisms --- p.22 / Chapter 1. --- Viable cell count --- p.22 / Chapter 2. --- Direct cell count --- p.22 / Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23 / Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23 / Chapter 1. --- Growth media and maintenance --- p.23 / Chapter 2. --- Culture Conditions --- p.24 / Chapter 3. --- Uptake of M. tuberculosis --- p.24 / Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25 / Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28 / Chapter 1. --- Primer and probe --- p.28 / Chapter 2. --- PCR conditions --- p.28 / Chapter 3. --- Detection --- p.29 / Chapter I. --- Patients and Clinical specimens --- p.30 / Chapter 1. --- Patients recruitment --- p.30 / Chapter 2. --- Clinical specimens --- p.30 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32 / Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32 / Chapter 2. --- Optimization of the PCR conditions --- p.32 / Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33 / Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34 / Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34 / Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35 / Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35 / Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35 / Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36 / Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36 / Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36 / Chapter 2. --- Pleural Fluid --- p.37 / Chapter 3. --- Tissue --- p.37 / Chapter 4. --- Sputum --- p.38 / Chapter 5. --- Cerebrospinal Fluid --- p.38 / Chapter X. --- DISCUSSION --- p.39 / Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39 / Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40 / Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41 / Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43 / Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43 / Chapter 2. --- Pleural fluids --- p.44 / Chapter 3. --- Tissues --- p.45 / Chapter 4. --- Sputa --- p.46 / Chapter 5. --- Cerebrospinal fluids --- p.46 / Chapter XI. --- CONCLUSION --- p.48 / Chapter XII. --- LITERATURE CITED --- p.50 / Chapter XIII --- TABLES --- p.67 / Chapter XIV. --- FIGURES --- p.85

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 107-114).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 141-164).

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 117-123.

This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis. Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM. Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' → 5' exonuclease activity of pol δ, displaying an IC50value greater than 1 mM. Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication. Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 169 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [114]-116).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 98-115).

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains x, 231 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 188-203).