by Lam Ying Hoo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 145-149). / Acknowledgements --- p.i / Abstract --- p.ii / Contents --- p.iv / Abbreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Trichosanthin --- p.1 / Chapter 1.2. --- Anti-plant viral and fungal properties of RIPs --- p.3 / Chapter 1.3. --- Agrobacterium-mediated transformation --- p.5 / Chapter 1.3.1. --- Ti (tumor inducing) plasmid --- p.6 / Chapter 1.3.2. --- Role of vir proteins in T-DNA transfer --- p.6 / Chapter 1.3.3. --- Integration of T-DNA into plant genome --- p.11 / Chapter 1.3.4. --- Use of Agrobacterium plasmid as transformation vectors --- p.13 / Chapter 1.4. --- Objective and strategy of producing transgenic plants that express TCS --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- Bacterial Strains used --- p.19 / Chapter 2.2. --- General Techniques --- p.20 / Chapter 2.2.1. --- Growth of bacterial strains --- p.20 / Chapter 2.2.2. --- Restriction Enzyme Digestion of DNA --- p.21 / Chapter 2.2.3. --- Agarose Gel Electrophoresis of DNA --- p.21 / Chapter 2.2.4. --- Purification of DNA fragments from Agarose Gel using GeneClean II® ( BIO 101 Inc.) kit --- p.22 / Chapter 2.2.5. --- Purification of DNA fragments by Phenol/Chloroform Extraction --- p.23 / Chapter 2.2.6. --- Ligation of DNA fragments --- p.24 / Chapter 2.2.7. --- Preparation and Transformation of Escherichia coli Competent Cells --- p.24 / Chapter 2.2.8. --- Minipreparation of Plasmid DNA --- p.26 / Chapter 2.2.9. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.27 / Chapter 2.2.10. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.29 / Chapter 2.2.11. --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.30 / Chapter 2.2.12. --- Western Blot detection of TCS --- p.33 / Chapter 2.2.13. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.3. --- Construction of Plant Transformation Vectors --- p.36 / Chapter 2.3.1. --- Construction of pSLJ58210 --- p.36 / Chapter 2.3.2. --- Construction of pSLJ TCS1 and pSLJ TCS2 --- p.38 / Chapter 2.3.3. --- Conjugation of pSLJ TCS1 and pSLJ TCS2 into A. tumefaciens by Triparental Mating --- p.41 / Chapter 2.4. --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens --- p.43 / Chapter 2.4.1. --- Growth of A. tumefaciens LBA4404 (pSLJ TCS1) --- p.43 / Chapter 2.4.2. --- Surface Sterilization of tobacco leaves --- p.43 / Chapter 2.4.3. --- Inoculation of tobacco leaf explants with A. tumefaciens LBA4404 (pSLJ TCS1) --- p.44 / Chapter 2.4.4. --- Regeneration of shoots from Transformed explants --- p.45 / Chapter 2.4.5. --- Rooting of Transformed shoots --- p.45 / Chapter 2.4.6. --- Re-establishment of cultured Plantlets in soil --- p.45 / Chapter 2.5. --- Analysis of the Regenerated Transgenic Tobacco --- p.46 / Chapter 2.5.1. --- Isolation of plant leaf protein --- p.46 / Chapter 2.5.2. --- SDS-PAGE and Western blot detection of TCS --- p.48 / Chapter 2.5.3. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.48 / Chapter 2.6. --- Bioassay of Inhibitory activity of TCS protein against TuMV --- p.49 / Chapter 2.6.1. --- Preparation of biologically active TCS protein --- p.49 / Chapter 2.6.2. --- Purification of TuMV from infected plant leaves --- p.51 / Chapter 2.6.3. --- Mechanical Inoculation of virus onto host plant --- p.53 / Chapter 2.6.4. --- Anti-viral assay on Local Lesion host --- p.54 / Chapter 2.6.5. --- Anti-viral assay on Systemic host --- p.55 / Chapter 2.7 --- Establishment of the plant culture medium for efficient Regeneration from tissue explants of Brassica parachinensis --- p.56 / Chapter 2.7.1. --- Preparation and Sterilization of culture medium --- p.57 / Chapter 2.7.2. --- Preparation of Sterile seedlings of B. parachinensis --- p.57 / Chapter 2.7.3. --- Regeneration from Cotyledon petiole and Hypocotyl segment explants --- p.58 / Chapter 2.7.4. --- Regeneration from Internode stem segment explants of shoot culture --- p.60 / Chapter 2.8. --- Reagents and Buffers --- p.61 / Chapter 2.8.1. --- Media for Bacterial culture --- p.61 / Chapter 2.8.2. --- Media for Plant tissue culture --- p.64 / Chapter 2.8.3. --- Restriction Enzymes --- p.66 / Chapter 2.8.4. --- Buffers for Agarose Gel Electrophoresis --- p.66 / Chapter 2.8.5. --- DNA ligation Buffer --- p.67 / Chapter 2.8.6. --- Reagents for preparation of E.coli competent cells --- p.67 / Chapter 2.8.7. --- Reagents for preparation of Plasmid DNA --- p.68 / Chapter 2.8.8. --- Reagents for Qiagen-pack 100 Cartridge --- p.69 / Chapter 2.8.9. --- Reagents for SDS-PAGE --- p.70 / Chapter 2.8.10. --- Reagents for Western Blotting --- p.71 / Chapter Chapter 3 --- Construction of Plant Transformation Vectors / Chapter 3.1. --- Introduction --- p.73 / Chapter 3.2. --- Results --- p.74 / Chapter 3.2.1. --- Construction of pSLJ58210 --- p.74 / Chapter 3.2.2. --- Construction of the recombinant binary vectors pSLJ TCSl and pSLJ TCS --- p.78 / Chapter 3.2.3. --- Conjugation ofpSLJ TCS 1 and pSLJ TCS 2 into Agrobacterium tumefaciens via Triparental Mating --- p.82 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Regeneration of leaf explants after transformation --- p.95 / Chapter 4.2.2. --- The level of expression of TCS in transgenic tobacco leaf --- p.100 / Chapter 4.3. --- Discussion --- p.104 / Chapter 4.3.1. --- Regeneration of transgenic tobacco plants --- p.104 / Chapter 4.3.2. --- Expression of TCS in transgenic tobacco plants --- p.108 / Chapter Chapter 5 --- Two approaches to study the Inhibitory effect of TCS on TuMV / Chapter 5.1. --- Introduction --- p.112 / Chapter 5.2. --- Results --- p.113 / Chapter 5.2.1. --- Expression and purification of recombinant TCS --- p.113 / Chapter 5.2.2. --- Purification of TuMV --- p.119 / Chapter 5.2.3. --- Anti-viral assay on local lesion host --- p.119 / Chapter 5.2.4. --- Anti-viral assay on Systemic host --- p.124 / Chapter 5.2.5. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.126 / Chapter 5.3. --- Discussion --- p.129 / Chapter Chapter 6 --- Establishment of plant culture conditions for efficient shoot regeneration from tissue explants of B.parachinensis / Chapter 6.1. --- Introduction --- p.133 / Chapter 6.2. --- Results --- p.133 / Chapter 6.3. --- Discussion --- p.137 / Chapter Chapter 7 --- Conclusion / Appendix / Chapter A.1. --- Size of molecular weight markers --- p.143 / Chapter A.2. --- References --- p.145

Leung Po-Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 204-235). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia - Aberrant Hematopoiesis --- p.4 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.4 / Chapter 1.1.2.2 --- Pathophysiology and Etiology of Leukemia --- p.7 / Chapter 1.1.2.3 --- Conventional Treatments for Leukemia --- p.9 / Chapter 1.1.2.4 --- New Avenues for Leukemia Therapy --- p.11 / Chapter 1.2 --- Coumarins: General Properties and Pharmacological Activities --- p.13 / Chapter 1.2.1 --- Introduction to Coumarins --- p.13 / Chapter 1.2.1.1 --- Historical Development of Coumarins --- p.13 / Chapter 1.2.1.2 --- Occurrence and Functions of Coumarins in Plants --- p.13 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.14 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.14 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.18 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.18 / Chapter 1.2.2.4 --- Metabolic Pathways and Pharmacokinetics of Coumarins --- p.19 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.22 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.22 / Chapter 1.2.3.2 --- Immunomodulatory Activity --- p.23 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.23 / Chapter 1.2.3.3.1 --- Mode of Entry of Coumarins into Tumor Cells --- p.23 / Chapter 1.2.3.3.2 --- Anti-carcinogenic Effect --- p.24 / Chapter 1.2.3.3.3 --- Anti-proliferative Activity --- p.25 / Chapter 1.2.3.3.4 --- Induction of Cell Differentiation --- p.26 / Chapter 1.2.3.3.5 --- Other Biological Activities --- p.26 / Chapter 1.2.4 --- Clinical Applications of Coumarins --- p.27 / Chapter 1.2.4.1 --- Treatment of Lymphoedema and Other High-protein Edemas --- p.27 / Chapter 1.2.4.2 --- Treatment of Thermal Injuries --- p.27 / Chapter 1.2.4.3 --- Therapeutic Agent for Renal Cell Carcinoma --- p.28 / Chapter 1.2.4.4 --- Therapy of Prostate Cancer --- p.29 / Chapter 1.3 --- Tumor Models Used in This Study --- p.30 / Chapter 1.3.1 --- Myeloid Leukemias --- p.30 / Chapter 1.3.1.1 --- HL-60 --- p.30 / Chapter 1.3.1.2 --- K562 --- p.30 / Chapter 1.3.1.3 --- EoL-1 --- p.30 / Chapter 1.3.1.4 --- WEHI-3B JCS --- p.31 / Chapter 1.3.2 --- Neuroblastoma - Neuro-2a BU-1 --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.33 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Cell Lines --- p.36 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.37 / Chapter 2.1.4 --- [methyl-3H] Thymidine (3H-TdR) --- p.40 / Chapter 2.1.5 --- Methylthiazoletetrazolium (MTT) --- p.41 / Chapter 2.1.6 --- Nitro Blue Tetrazolium (NBT) --- p.41 / Chapter 2.1.7 --- Liquid Scintillation Cocktail --- p.42 / Chapter 2.1.8 --- Reagents and Buffers for Flow Cytometry --- p.42 / Chapter 2.1.9 --- Mouse Anti-MAP-2 Monoclonal Antibody --- p.44 / Chapter 2.1.10 --- Reagents for DNA Extraction --- p.44 / Chapter 2.1.11 --- Reagents for Total RNA Isolation --- p.45 / Chapter 2.1.12 --- Reagents and Buffers for RT-PCR --- p.46 / Chapter 2.1.13 --- Reagents and Buffers for Gel Electrophoresis --- p.49 / Chapter 2.1.14 --- Reagents and Buffers for Western Blot Analysis --- p.50 / Chapter 2.1.15 --- Reagents for Measuring Caspase Activity --- p.58 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.61 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.61 / Chapter 2.2.3 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.62 / Chapter 2.2.4 --- Determination of Cell Viability --- p.62 / Chapter 2.2.5 --- Cell Morphology Study --- p.63 / Chapter 2.2.6 --- Immunocytochemistry --- p.64 / Chapter 2.2.7 --- Confocal Microscopy --- p.64 / Chapter 2.2.8 --- NBT Reduction Assay --- p.65 / Chapter 2.2.9 --- In vivo Tumorigenicity Assay --- p.65 / Chapter 2.2.10 --- In vivo Anti-tumor Study --- p.65 / Chapter 2.2.11 --- Measurement of In vivo Macrophage Migration --- p.66 / Chapter 2.2.12 --- Measurement of Cytokine Production by ELISA --- p.66 / Chapter 2.2.13 --- Measurement of Apoptosis by DNA Fragmentation Analysis --- p.67 / Chapter 2.2.14 --- Determination of the Mitochondrial Membrane Potential --- p.67 / Chapter 2.2.15 --- Cell Cycle/DNA Content Evaluation --- p.68 / Chapter 2.2.16 --- Nitric oxide/Annexin V-PE Dual Sensor Assay --- p.68 / Chapter 2.2.17 --- Gene Expression Study --- p.68 / Chapter 2.2.18 --- Protein Expression Study --- p.71 / Chapter 2.2.19 --- Measurement of Caspase Activity --- p.74 / Chapter 2.2.20 --- Statistical Analysis --- p.75 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTIVITIES OF COUMARINS ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Results --- p.18 / Chapter 3.2.1 --- Differential Anti-proliferative Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.78 / Chapter 3.2.2 --- Cytotoxic Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.91 / Chapter 3.2.3 --- "Kinetic, Reversibility and Stability Studies of the Anti- proliferative Effect of Coumarins on the Leukemia JCS cells" --- p.94 / Chapter 3.2.4 --- Induction of DNA Fragmentation in Myeloid Leukemia Cells by Coumarins --- p.100 / Chapter 3.2.5 --- Effect of Coumarins on the Cell Cycle Kinetics of the Leukemia JCS Cells In Vitro --- p.107 / Chapter 3.2.6 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Leukemia JCS Cells --- p.112 / Chapter 3.2.7 --- Effect of Esculetin on the In Vivo Growth of the Leukemia JCS cells in Syngeneic Mice --- p.115 / Chapter 3.3 --- Discussion --- p.117 / Chapter CHAPTER 4: --- AN INVESTIGATION ON THE DIFFERENTIATION- INDUCING EFFECT OF COUMARINS / Chapter 4.1 --- Introduction --- p.122 / Chapter 4.2 --- Results --- p.124 / Chapter 4.2.1 --- The Differentiation-inducing Effect of Coumarins on Myeloid Leukemia Cells --- p.124 / Chapter 4.2.1.1 --- Morphological Changes in Coumarin-treated HL-60 Cells --- p.124 / Chapter 4.2.1.2 --- NBT Reduction of HL-60 Cells --- p.127 / Chapter 4.2.1.3 --- Effects of Coumarins on the Cell Size and Granularity of HL-60 Cells --- p.129 / Chapter 4.2.2 --- The Anti-proliferative and Differentiation-inducing Effects of Coumarins on Neuroblastoma Cells --- p.131 / Chapter 4.2.2.1 --- Anti-proliferative Effect of Coumarins on the BU-1 Cell Line In Vitro --- p.131 / Chapter 4.2.2.2 --- Morphological Changes in Coumarin-treated BU-1 Cells --- p.134 / Chapter 4.2.2.3 --- Immunocytochemistry of Coumarin-treated BU-1 Cells --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI-LEUKEMIC ACTIVITIES OF COUMARINS / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Genes in the Leukemia JCS Cells --- p.147 / Chapter 5.2.2 --- Modulatory Effects of Coumarins on the Expression of Growth-related Genes in the Leukemia JCS Cells --- p.151 / Chapter 5.2.3 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Proteins in Leukemia JCS Cells --- p.157 / Chapter 5.2.4 --- Modulatory Effects of Coumarins on the Expression of Growth-related Proteins in Leukemia JCS Cells --- p.162 / Chapter 5.2.5 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Leukemia JCS cells --- p.165 / Chapter 5.2.6 --- Induction of Apoptosis and Nitric Oxide Production in Leukemia JCS Cells by Coumarins --- p.168 / Chapter 5.2.7 --- Effects of Coumarins on the Caspase Activity in the Leukemia JCS cells --- p.172 / Chapter 5.3 --- Discussion --- p.177 / Chapter CHAPTER 6: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES / Chapter 6.1 --- Introduction --- p.185 / Chapter 6.2 --- Results --- p.188 / Chapter 6.2.1 --- Effect of Coumarins on the Viability of Macrophages In vitro --- p.188 / Chapter 6.2.2 --- Effect of Coumarins on the In vivo Migration of Macrophages --- p.190 / Chapter 6.2.3 --- Effect of Coumarins on Cytokine Production by Macrophages --- p.192 / Chapter 6.3 --- Discussion --- p.194 / Chapter CHAPTER 7: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.197 / REFERENCES --- p.204

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

<p>The aim of the study is to determine the characteristics associated with ART adherence to , in Albert Horsfall Medicacal Center (AHMC), Abuja, Nigeria. The Objectives are to measure the adherence to ART, assess the association of the demographic characteristic of patients on ART with adherence to medications in the facility / and to establish patients' perspectivesto adherence and impediments to compliance to ART in the center.</p>

Context: Suboptimal drug use is a major contributor to adverse patient outcomes in primary care. Considering their accessibility and frequent interactions with patients, community pharmacists may be well suited to identifying patients who are at high risk of drug therapy problems (DTPs) and who may benefit from a comprehensive medication assessment. Objective: To determine if a short screening tool can identify patients at risk for DTPs in a community pharmacy setting. Design: A five question self-administered screening tool was identified in the literature and adapted to reflect current practice in community pharmacy. Adults requesting a refill prescription from three different community pharmacies over 12 weeks completed the screening tool, and had a comprehensive medication assessment with a pharmacist. Information from the assessment was used to: a) determine the ability of patients to correctly answer the screening tool questions and to classify themselves into the appropriate risk category (High or Low Risk); b) compare the number of DTPs identified in each risk category (High vs Low); and c) determine the number of High Risk and Low Risk patients who would qualify for any of the existing provincial medication review programs in Canada. Results: 49 patients completed the study. Most patients were able to answer the questions on the screening tool correctly. The strength of agreement was very good (Kappa 0.91, p<0.01) between the overall patient determined risk category and pharmacist determined risk category. Patients identified as High Risk (n=18) had a mean of 3.72 (p<0.01) more DTPs than Low Risk patients (n=31). All but one (94.4%) of the High Risk patients had at least one Moderate or Severe DTP, while less than half (48.4%) of Low Risk patients had at least one Moderate or Severe DTP. The majority of High Risk patients were eligible for medication reviews in all programs except for Newfoundland and Labrador, New Brunswick and Saskatchewan. Close to a third of Low Risk patients were eligible for medication reviews in Prince Edward Island, Nova Scotia and Ontario. Conclusions: This screening tool is a trustworthy method for identifying patients in community pharmacies who have a large number of DTPs. Patients identified as High Risk using this screening tool may be good targets for community pharmacy based comprehensive medication assessments.

<p>The aim of the study is to determine the characteristics associated with ART adherence to , in Albert Horsfall Medicacal Center (AHMC), Abuja, Nigeria. The Objectives are to measure the adherence to ART, assess the association of the demographic characteristic of patients on ART with adherence to medications in the facility / and to establish patients' perspectivesto adherence and impediments to compliance to ART in the center.</p>

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thesis (M.S.)--University of Wisconsin--Madison, 1990. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 72-84).

Magister Public Health - MPH / The aim of the study is to determine the characteristics associated with ART adherence to , in Albert Horsfall Medicacal Center (AHMC), Abuja, Nigeria. The Objectives are to measure the adherence to ART, assess the association of the demographic characteristic of patients on ART with adherence to medications in the facility; and to establish patients' perspectivesto adherence and impediments to compliance to ART in the center. / South Africa