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Of the considerable challenges researchers face in the control and elimination of malaria, the development of antimalarial drug resistance in parasite populations remains a significant hurdle to progress worldwide. Atovaquone is used in combination with proguanil (Malarone) as an antimalarial treatment in uncomplicated malaria, but is rendered ineffective by the rapid development of atovaquone resistance during treatment. Previous studies have established that de novo mutant parasites confer resistance to atovaquone with a substitution in amino acid 268 in the cytochrome b gene encoded by the parasite mitochondrial genome, yet much is still unknown about how this resistance develops, and whether parasites are inherently predisposed to resistance development. Here we report phenotypic characterization of isolates from patients that failed treatment in the original atovaquone Phase II studies in Thailand by using a diverse series of chemotypes that target mitochondrial functions. We defined their structure-activity relationships and observed broad resistance (5-30,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient to explain this spectrum of resistance. We also report the first known in vitro selection that recapitulates the clinical Y268S mutation using the TM90-C2A genetic background, the pre-treatment parent for TM90-C2B. Selection of the Y268S mutation in TM90-C2A and others indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other genetic backgrounds results in mutations at positions other than amino acid 268. We implicate mitochondrial heteroplasmy in the development of sporadic, rapid resistance to atovaquone, where pre-existing low-level mutations in the multi-copy mitochondrial DNA can be quickly selected for in parasite populations. High-coverage mitochondrial deep-sequencing data showed that low-level Y268S mutants were present in admission parasites from the atovaquone Phase II clinical trials in Thailand, and recrudescent parasites either maintained high level Y268S mutation frequencies or gradually returned to cryptic Y268S levels. The phenomenon of gradual heteroplasmic conversion back to wild-type was noted in some ex vivo patient isolated parasites as well as some in vitro selected lines, which suggests that other factors are at play that influence heteroplasmy stability. In addition to mitochondrial heteroplasmy, the total mtDNA copy number is likely influencing phenotypes in a gene dose-dependent fashion. Further, pressure on the DHODH enzyme that results in DHODH copy number amplifications/mutations has been shown to influence mitochondrial heteroplasmy directly. Last, mitochondrial diversity was shown to be vastly underestimated without heteroplasmic loci being taken into account, as seen in the re-analysis of the Pf3K MalariaGEN genome dataset we performed. The complex interactions between these drug resistance mechanisms reveal the phenotypic and genotypic plasticity that the Plasmodium falciparum parasite utilizes are a clear fitness advantage in the face of mitochondrial stress, and further studies are required to determine the impact of this wide-ranging phenotype on the development of new mitochondria-targeted drugs.

Diarrhea is a global concern that the United Nations Children’s Fund (UNICEF) and the World Health Organization (WHO), have confirmed to be the second major cause of death in children under the age of five. Major bacterial pathogens that cause diarrhea include Escherichia coli, Salmonella species, Shigella species and Staphylococcus aureus. Antibiotic therapy is recommended depending on the severity and presentation of the disease; however, the appearance of antibiotic-resistant bacteria is an emerging global threat to the ability to treat these bacterial infections. This situation could be overcome by the discovery of new natural antibiotics. Plants have been a source of medicine for centuries and have been used to treat diseases including diarrhea. This makes plants a natural potential target to study for their antibiotic properties. The objective of this study was to determine the antibiotic properties of medicinal plants against known pathogens that cause bacterial diarrhea. Three medicinal plants, Cassia abbreviata, Kigelia africana, and Geranium incanum were investigated for their antimicrobial properties against these strains of microorganisms: American Type Culture Collection (ATTC) and Clinical Strains (CS). The plant materials were ground into powder, which was then dissolved in methanol, acetone and distilled water to extract the active compounds. The plant extracts were then used to (i) determine their antibiotic activity, (ii) determine the minimum inhibitory concentration (MICs), (iii) analyze the thin layer chromatography (TLC) fingerprints, and (iv) analyze the autobiography assay. The results obtained in this study met the aim and objectives of this study. The antimicrobial activities of the selected plants were obtained as discussed in Chapter 2 and 3. These results indicated that the traditional plants could be used as antimicrobials. In the screening assays, the test microorganisms were inhibited by the plant extracts, when they were subjected to plant extracts. This was performed on Mueller Hinton agar as sensitivity testing, which revealed clear zones of inhibition. The MIC values for each plant extract were established which ranged from 0.101 to 13.3 mg/dl. The TLC analysis revealed the spots which contained the active compounds which inhibited the bacterial growth. A bioautography assay was performed on the TLC plates, which exposed the exact spots containing the active compound inhibiting the bacteria. These results are clearly consistent with what former scientists have observed. Detailed explanations on the results are in Chapter 3 and 4 of this paper. It is important to note that all the procedures performed in this study were in vitro assays. Some effective in vitro assay activity may not always result in the same effective in vivo activity, because some active compounds may be metabolized and degraded into inactive metabolites. For this reason, the in vitro results obtained in this study, may not reflect the true effectiveness of the compounds in in vivo trials. It is therefore advised that future scientists should take a step further in analyzing the plant extracts through in vivo assays. Further testing and study on these plants at an advanced molecular level will be beneficial in the medical fields in the search for new antibiotics to treat infectious diseases. Purification and further analysis of their products can be helpful in the production of pure natural medicines. This will discover the active ingredients and compounds responsible for inhibition of the microorganisms. This will make the compounds potential candidates for a scientific validation and analysis for future scientists to bring a new dawn in the fight against infectious diseases.

The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.

Helicobacter pylori classified as a class 1 carcinogen is a common human pathogen implicated in certain gastrointestinal diseases. Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries. H. pylori infection causes peptic ulcer, duodenitis, gastritis and cancer. The growing resistance of H. pylori to antibiotics used in its treatment as well as other innate limitations of the triple therapy has necessitated a search for alternative treatment from natural sources which could be readily available, less cost effective. The antimicrobial activity of solvents (acetone, ethanol, methanol, chloroform and water) crude extracts of Lippia javanica were investigated against 31 H. pylori strains by the agar well diffusion technique. The minimum inhibitory concentration (MIC) was determined by spectrophotometric analysis at 620 nm using the broth micro dilution method and the rate of kill by broth dilution method. Phytochemical analysis was also performed. H. pylori standard strain NCTC 11638 was included as a positive control. Metronidazole and amoxicillin were used as positive control antibiotics. The ANOVA test was used to analyze the results using SPSS version 17.0. The strains were inhibited by all the extracts with inhibition zones of diameter ranging from 0-36 mm and 0-35 mm for the control antibiotic, clarithromycin. The MIC90 ranged from 0.039- 0.625 mg/mL for acetone; 0.039-1.25mg/mL for methanol, 0.00195-0.313 mg/mL for ethanol; 0.01975-2.5 mg/mL for metronidazole and 0.0048-2.5 mg/mL for amoxicillin. Acetone extract completely inhibited strain PE369C at MIC (0.1 mg/mL) and 2× MIC (0.2 mg/mL) in 18h and at ½× MIC (0.05 mg/mL) in 36h. Strain PE466C was completely inhibited at 4× MIC in 72h. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids and alkaloids. The results indicate that the extracts of the leaves of L. javanica may contain compounds with anti-H. pylori activity and merits further study to identify the compounds.

Continuous-time Markov chains (CTMC's) form a convenient mathematical framework for analyzing random systems across many different disciplines. A specific research problem that is often of interest is to try to predict maximum probability sequences of state transitions given initial or boundary conditions. This work shows how to solve this problem exactly through an efficient dynamic programming algorithm. We demonstrate our approach through two different applications - ranking mutational pathways of HIV virus based on their probabilities, and determining the most probable failure sequences in complex fault-tolerant engineering systems. Even though CTMC's have been used extensively to realistically model many types of complex processes, it is often a standard practice to eventually simplify the model in order to perform the state evolution analysis. As we show here, simplifying approaches can lead to inaccurate and often misleading solutions. Therefore we expect our algorithm to find a wide range of applications across different domains.

Human health is increasingly threatened by the emergence of multiply drug resistant malignant organisms. Yet, our understanding of the numerous ways by which such resistance arises is modest. Here, we present evidence of a bet hedging strategy in the budding yeast, Saccharomyces cerevisiae, to counter the effects of cytotoxic drugs through the action of Pdr5, an ATP-binding cassette transporter. We have employed flow cytometry and fluorescent activated cell sorting to probe the expression levels of a GFP-tagged version of PDR5 in individual cells. The results obtained from these experiments demonstrate that each yeast population is variable in the levels of Pdr5 production, and a small subpopulation of cells produces this efflux pump at much higher quantities than the population average. Consequently, cells with high and low levels of Pdr5 grow differentially in presence and absence of cycloheximide, a cytotoxic drug. These properties are highly suggestive of a bet hedging strategy mediated by Pdr5 levels. We further link this bet hedging strategy to the transcriptional regulatory network of PDR5 consisting of two major transcription factors, Pdr1 and Pdr3. Our analysis suggests that a self-activating feedback loop acting on Pdr3 plays an important role in generation of the aforementioned subpopulation. Furthermore, our results point to a large difference in the activity of these two regulators wherein Pdr3 is notably stronger than Pdr1. The disparity in their activity could indicate a mechanism for generation of the observed proportions of subpopulations with regards to the level of Pdr5.

Since heparin combines with some antibiotics to decrease the toxicity of the antibiotic to the patient, the purpose of this investigation is to determine whether it has any effect upon the development of resistance to antibiotics by Staphylococcus aureus.

The full text of this thesis/dissertation is not available online. Please <a href="mailto:upetd@up.ac.za">contact us</a> if you need access. Read the abstract in the section 00front of this document. / Dissertation (MSc (Medical Immunology))--University of Pretoria, 2006. / Immunology / unrestricted

Thirty-one rats were trained on a differential reinforcement of low rate schedule. After responding had stabilized, animals were injected with methylphenidate, twice weekly, presession. Methylphenidate produced dose-dependent increases in response rates and decreases in reinforcements. Repetition of these doses produced a reduced drug effect, and a third administration of the 10 mg/kg dose further reduced the drug effect. Subsequently, the effects of daily and intermittent administration were determined for this dose. Daily methylphenidate, pre-session, produced tolerance to the behavioral effects of methylphenidate and cross-tolerance to the amphetamines. Twice-weekly methylphenidate, pre-session, produced partial tolerance to methylphenidate and partial cross-tolerance to the amphetamines. Thus, periodic exposure to the behaviorally disruptive effects of a drug of the amphetamine class reduces the effects of subsequent exposure.