Haemonchus contortus is a nematode parasite that infects sheep and goats, and other ruminants, causing substantial economic loss throughout the world. Anthelmintic drugs are the primary method to control these parasites; however, resistance to all broad spectrum anthelmintics, including the newer avermectins and milbemycins, has been developing rapidly in nematode parasites. The mechanism of avermectin resistance is unknown, but previous studies indicate that at least four genes are involved. A single nucleotide polymorphism (SNP) in the Haemonchus contortus HG1 gene has been linked to ivermectin (IVM) and moxidectin (MOX) resistance. This gene encodes a GABA-gated chloride channel thought to control body muscle movement in H. contortus and mediates IVM and MOX paralysis. The present study was designed to determine whether there is an association between variation in this gene and the ability of H. contortus larvae to move in the presence and absence of MOX. The present study also investigated the difference between a laboratory strain (PF23) and an ivermectin resistant field strain (VHR29). Fourteen different MOX concentrations (ranging from 0.078nm to 156uM) and at least 200 third stage (L3) larvae for each concentration were investigated for each strain. An L3 larval motility assay was used to determine the movement phenotype of individual larva. Pyrosequencing was used to identify the genotype of each individual larva. The present study found that in the PF23 strain, the resistant heterozygote genotype GA protected against the effects of MOX, and that the protection was dose dependent. This was not the case, however, in the VHR29 field strain. For this strain, no protective effect of the HG1 variation was observed with moxidectin treatment. One reason for this may be that field strains are selected under higher drug pressure which favors a monogenic response. The present study has shown that while the HG1 variation does play a role in larval movement and resistance to MOX, this effect may be minor compared to other effects selected in the field.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

The development of intratumoral hypoxia is associated with resistance to therapy in many forms of human cancer, and pre-exposure of tumor cells to hypoxia confers multidrug resistance. Research over the last several years has led to considerable advances in the understanding of the cellular response to oxygen deprivation, however the hypoxia-induced mechanisms that contribute to the chemoresistance phenotype are still not well understood. Recent studies have identified hypoxia-inducible factor 1 (HIF-1), a master transcriptional regulator of oxygen homeostasis, as an important mediator of hypoxia-induced chemoresistance in cancer cells. The research described in this thesis confirms these findings and demonstrates HIF-1 is required for hypoxia-induced resistance to doxorubicin and etoposide in human tumor cells. In addition, novel findings revealed that hypoxia-induced drug resistance occurred independently of changes in the apoptotic fraction and was associated with decreased drug-induced senescence. DNA damage measured at the single-cell level revealed that the increase in survival correlated well with a HIF-1-dependent decrease in etoposide-induced DNA strand breaks, providing direct evidence that exposure of tumor cells to hypoxia leads to protection against some forms of drug-induced DNA damage. Characterization of several classical mechanisms of drug resistance upstream of DNA damage identified multiple determinants of cellular resistance to anticancer agents. The relative contributions of each varied depending on the particular drug and cancer cell line studied. Together, the findings presented here support a model in which hypoxia-induced chemoresistance is a multifactorial phenomenon that is regulated, at least in part, through HIF-1-dependent mechanisms. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-08-29 12:35:36.219

The objectives of this study are to estimate the degree to which a glutamate-gated chloride channel gene (HcGluCla) contributes to survival of moxidectin treatment and to study the relative dominance of those alleles. The phenotype of individual adult H. contortus with respect to feeding was determined using an inulin uptake assay. Genotype was determined using a diagnostic PCR assay. In the absence of moxidectin, homozygous susceptible genotypes fed significantly more than homozygous resistant genotypes. The effect of the susceptible allele was dominant. In the presence of moxidectin, feeding in the susceptible homozygotes was reduced to the level found in the resistant homozygotes, which were unaffected by the drug. These results suggest that the function of the two alleles is different and that they also respond differently to the drug, the resistant allele being unaffected by the drug. / The selection coefficient, s, is the selective difference between the resistant and susceptible genotypes with regard to feeding. Parasites with the resistant allele were seen to feed less in the absence of the drug, i.e., the effect is recessive. In the presence of the drug, there was no difference between resistant and susceptible parasite feeding. These results suggest that resistance may have hidden complexities. (Abstract shortened by UMI.)

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

The anti-nematodal drug ivermectin hyperactivates invertebrate-specific glutamate-gated chloride channels (GluCls) causing pharyngeal paralysis and a cessation of feeding and growth. I find that for C. elegans even brief exposure to ivermectin can lead to irreversible pharyngeal paralysis. Ivermectin induces heterogeneous vacuolation in the pharynx that appears slowly and accumulates over several days. This vacuolation is almost completely rescued by a mutation in avr-15, which codes for the alpha-subunit of pharyngeal GluCls. The vacuoles stain strongly with Lysotracker Red and are therefore likely to be acidic compartments of the endosomal-lysosomal system. Examination of mutants defective for endocytosis (rme-1, rme-8, and cup-5) uncovers the presence of acidic vacuoles identical in appearance to ivermectin-induced vacuoles. Further, RME-1, a marker for recycling endosomes, is shown to redistribute soon after ivermectin exposure. Examination of the effects of ivermectin on extrapharyngeal neurons expressing ectopic avr-15 reveals an apoptotic phenotype that is shown to be ced-independent.

As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.

Pyrimethamine efficacy was investigated in vitro on the blood asexual stages, the sexual stages and exflagellation in Plasmodium falciparum. Gametocytogenesis was stimulated following the standard methods on five isolates of Plasmodium falciparum. From these five isolates, RSA 2, 3 and 5 produced gametocytes which reached maturity within seven days and the gametocytes were able to exflagellate. Isolate MW2 produced young gametocytes which disappeared within ten days. NF54 produced mature gametocytes which lasted for 24 hours only. There were no statistically significant differences between the static and the synchronization methods of gametocyte stimulation for any of the isolates. The effect of pyrimethamine was investigated by adding a known concentration of the drug (For RSA 2, MW2 and NF54, l00nmol/ℓ; RSA 3 and 5, 3000nmol/ℓ pyrimethamine) to the culture medium for seven days during gametocyte stimulation. The results of this investigation show that there was gametocytocidal activity on the isolates that were used and pyrimethamine also had a schizontocidal action on NF54 and the young gametocytes of this isolate were destroyed by the drug. At concentrations which were inhibitory to asexual parasites, the drug had a sporontocidal effect on isolate RSA 2 but not on isolate RSA 5. The pyrimethamine MIC values for asexual parasites ranged from 300nmol/ℓ to > 3000nmol/ℓ (RSA 2 and 5 were not inhibited at 3000nmol/ℓ ). These results are consistent with those found in previous studies when pyrimethamine resistance was first detected in South Africa. The chloroquine MICs indicate a good correlation with the results obtained from previous drug sensitivity tests for all the isolates examined using both the 48-hour in vitro test and isotope incorporation for growth assessment. The isobolograms constructed to determine relationship between chloroquine and pyrimethamine indicated no synergism for isolates RSA 2 and 5, but the Σ relative IC[50]s indicated a weak synergism. Both the isobolograms and the Σ relative IC[50]s for the isolates RSA 6, 9 and 14 indicated an antagonistic action between chloroquine and pyrimethamine. The results obtained from this study have important implications for malaria control in South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.