Magister Public Health - MPH / HIV/AIDS is a major health problem in Namibia with HIV prevalence estimated at 18.2% among pregnant women. Antiretroviral therapy (ART) was introduced in the public sector in 2003 and ART roll out was expanded throughout the country in the subsequent years. There are 221 ART sites in Namibia which include 34 district hospitals and 187 outreach service points. Currently there are 127,486 patients registered on ART in Namibia. However, there have been cases of patients experiencing treatment failure. The treatment failure can give rise to the emergence of HIV drug resistance. Genotyping information from patients with treatment failure can be valuable for tracking the dominant mutations conferring HIV drug resistance. However, HIV genotyping is not routinely available in Namibia due to cost. It is essential to determine the risk factors associated with development of HIV drug resistance so that these factors can be addressed. The aim of the current study was to describe HIV drug resistance mutations and the risk factors associated with HIV drug resistance among patients failing first- line ART regimen in Oshikoto and Oshana regions in Namibia. The case-control study design was used to collect data from cases who were being suspected of treatment failure to the first–line regimen in Oshikoto and Oshana regions in Namibia. The demographic, clinical and genotype information was collected from patient records. Out of 168 cases, 97 cases were eligible for this study and were matched with 105 controls. The mean age was 44.8 (±13.2) years for controls and 43.3 (±13.3) years for cases. Cases from Oshana and Oshikoto regions harboured 63% and 71% respectively for nucleoside reverse transcriptase inhibitors mutations with the dominant mutation being M184V/I. Sixty-eight percent (68%) and 76% respectively harboured mutations for non-nucleoside reverse transcriptase inhibitors with dominant mutation being K103N. Missed appointments, initiating inappropriate first-line regimen and adverse events or side effects were identified as risk factors for virological failure with odd ratios (OR) of 21.58 (95% CI 6.50 -71.59); 11.70 (95% CI 1.69 - 80.99) and 7.17 (95% CI 1.89 -27.22) respectively. Patients failing the first-line regimen need to be genotyped to assess the development of HIV drug resistance. The patients initiating ART should be educated on impacts of missing clinical appointments and adverse events of the drugs in order to prevent the emergence of drug resistance.

Malaria remains a debilitating disease, especially in developing countries of the tropics and sub-tropics. Increasing drug resistance and the rising cost of drug development calls for methods that can cost-effectively identify new drugs. The proteins of the malaria causing Plasmodium parasites often exhibit unique features compared to their mammalian counterparts. Such features invite discovery of parasite-specific drugs. In this study computational methods were applied to understand unique structural features of enzymes from the Plasmodium polyamine biosynthesis pathways. Molecular modeling of P. falciparum arginase was used to explore the structural metal dependency between enzyme activity and trimer formation. This dependency is not observed in the mammalian host. A novel inter-monomer salt-bridge was discovered between Glu 295 and Arg 404 that helps mediate the structural metal dependency. Removal of the active site metal atoms promoted breaking of the Glu 295á::Arg 404b interaction during simulation. The involvement of this salt-bridge was further confirmed by site-directed mutagenesis of the recombinantly expressed enzyme and subsequent simulation of the mutants in silico. Mutations designed to break the salt-bridge resulted in decreased enzyme activity and oligomerisation. Furthermore, simulation of the mutants indicated potential loss of metal co-ordination within the active site. The interface around Glu 295á::Arg 404b could thus serve as a novel therapeutic target. In Plasmodium the usually separate activities S-adenosylmethionine decarboxylase and ornithine decarboxylase occur in a single bifunctional enzyme. Previous studies have established the importance of complex formation and protein-protein interactions for correct enzyme functioning. Disturbing these interactions within the complex may therefore have inhibitory potential. In the second aspect of this study the potential quarternary structure of AdoMetDC/ODC was studied by homology modeling of the domains followed by protein-protein docking. The results from five Plasmodium species suggest that one face of each domain is favoured for complex formation. The predicted faces concur with existing experimental results, suggesting the direct involvement of Plasmodium-specific inserts in maintaining complex formation. Further fine-grained analysis revealed potentially conserved residue pairs between AdoMetDC/ODC that can be targeted during experimental follow-up. In both aspects of this study computational methods yielded useful insights into the parasite-specific features of polyamine biosynthesis enzymes from Plasmodium. Exploitation of these features may lead to novel parasite-specific drugs. Furthermore, this study highlights the importance of simulation and computational methods in the current and future practice of Science. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

The antibacterial potency of the extracts of the seed of Garcinia kola (bitter kola) was investigated in this study against a panel of referenced, environmental and clinical bacterial strains. The killing rates of the active extract as well as their potential for combination antibacterial therapy with standard antibiotics were also elucidated using standard procedures. The aqueous and acetone extracts of the seed were screened for activity against 27 bacterial isolates. The aqueous extract exhibited activity mainly against Gram positive organisms with Minimum inhibitory concentration (MIC) values ranging from 5 mgml-1 – 20 mgml-1, while the acetone extract showed activity against both Gram negative and Gram positive organisms with MIC values ranging from 10 mgml-1 - 0.156 mgml-1. The acetone extract also showed rapid bactericidal activity against Staphylococcus aureus ATCC 6538 with a 3.097 Log10 reduction in counts within 4 hours at 0.3125 mgml-1 and a 1.582 Log10 reduction against Proteus vulgaris CSIR 0030 at 5 mgml-1 after 1 hour. In addition, the aqueous, methanol and acetone extracts of the seeds also exhibited activity against four clinical strains of Staphylococcus isolated from wound sepsis specimens. The MIC values for the aqueous extract were 10 mgml-1 for all the isolates while the acetone and methanol extracts had lower values ranging from 0.3125 - 0.625 mgml-1. The acetone extract was strongly bactericidal against Staphylococcus aureus OKOH3 resulting in a 2.70 Log10 reduction in counts at 1.25 mgml-1 within 4 hours of exposure and a complete elimination of the organism after 8 hours. The bactericidal vi activity of the same extract against Staphylococcus aureus OKOH1 was weak, achieving only a 2.92 Log10 reduction in counts at 1.25 mgml-1 (4× MIC) in 24 hours. In the test for interactions between the acetone extract of the seeds and antibiotics, synergistic interactions were observed largely against Gram positive organisms using the FIC indices, (indices of 0.52 - 0.875) with combinations against Gram negatives yielding largely antagonistic interactions (indices of 2.0 to 5.0). Synergy (≥ 1000 times or ≥ 3 Log10 potentiation of the bactericidal activity) against both Gram negative and Gram positive organisms was detected by time kill assays mainly involving the antibiotics tetracycline, chloramphenicol, amoxycillin and penicillin G. Combinations involving erythromycin and ciprofloxacin consistently gave antagonistic or indifferent interactions. We conclude that the acetone extract of Garcinia kola seeds possess strong bactericidal activities against both Gram positive and Gram negative organisms and can be therapeutically useful in the treatment of bacterial infections including the problematic staphylococcal wound infections. In addition, the acetone extract can be a potential source of broad spectrum resistance modifying compounds that can potentially improve the performance of antibiotics in the treatment of drug resistant infections.

A dissertation submitted to Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine, Johannesburg, June 2018 / It is well known that antiretroviral (ARV) drug resistant variants of HIV-1 can be sexually transmitted. Several studies have shown that in resource-rich geographical locations as many as 15-20% of individuals are newly infected with HIV-1 containing at least one drug resistant mutation. In contract, resource limited geographical locations, such as Sub-Saharan Africa, have shown prevalences in the range of 5 to 10%. Since the ART rollout in these resource-limited locations are generally not well monitored with virological genotyping, the transmission of drug resistant HIV-1 is likely to increase, with significant clinical and public health consequences. HIV-1 transmission is characterised by the transmission of a single founder virus, or narrow spectrum of founder viruses, that develop into the viral quasispecie. It is unlikely that drug resistant virus will coexist with wild type (wt) virus, in the case of non-drug resistance transmission. However, initiating in ARV treatment, drug non-adherence may select of ARV drug resistance mutations and may subsequent lead to treatment failure. Drug resistant virus may be transmitted to a new host, as drug resistant mutations do not appear to hamper transmission efficiency of the mutated virus. Several studies have shown that transmitted drug resistance mutations (TDRMs) persist either as the dominant species or as minority variants, or revert to wild type over time, in the absence of drug pressure. It is generally acknowledged that many drug resistance mutations decrease the replicative capacity of HIV-1, and thus reversion confers a potential survival advantage. Because of the emergence of wild type variants from TDRM quasispecies requires evolution and back-mutation, the rate at which individual TDRMs become undetectable may vary substantially. Contradictory findings of persistence versus reversion of TDRMs have been reported, and may be attributed to the fact that minority variants are difficult to detect by conventional population based Sanger sequencing, and patient numbers studied are small. Consequently, individuals infected with HIV-1 harbouring TDRM have a higher chance of failing their first-line therapy. Understanding the population dynamics of transmitted drug resistant HIV-1 in the absence of drug pressure is essential for clinical management and public health strategies. The individuals identified with TDRMs from the IAVI-Early Infections Cohort (Protocol C) provides a unique research opportunity to address the aforementioned issue. This study describes III the evolutionary mechanisms of ARV drug resistant HIV-1 after transmission to a new host to provide insight into persistence and/or rates of reversion to wild type. TDRMs initially identified by Price et al. (2011) in the IAVI-Early Infections Cohort (Protocol C) using population-based Sanger sequencing (the current diagnostic gold standard), were confirmed in this study by newer ultra-deep next generation sequencing (NGS) technology on the Illumina Miseq platform. Longitudinal samples were made available for individuals in which transmitted drug resistance were identified, and we also sequenced using NGS on the Illumina Miseq platform. Additional minority variants (present at <20% of the sequenced viral population) were identified by NGS. This study found a large percentage of TDRMs to persist for a significant amount of time after transmission to a new, drug naïve host, in the longitudinal samples. The level of persistence, or rate of reversion of TDRMs, appear to be subject to the type of resistance (NRTI, NNRTI or PI), level of resistance the mutation confers, as well as the combination of mutations that are cotransmitted. Findings of this study highlight the importance of drug resistance screening prior to ART initiation, as well as the importance of the drug resistance screening assay sensitivity. As rates of transmitted drug resistance are increasing in developing countries of which the IAVI-Early Infections Cohort (Protocol C) are composed of, understanding the population dynamics of transmitted drug resistant HIV-1 in the absence of drug pressure is essential for clinical management, public health strategies and informing future vaccine design. / XL2018

Liposomal doxorubicin is a clinically important drug formulation indicated for the treatment of several different forms of cancer. For doxorubicin to exert a therapeutic effect, it must gain access to the nucleus. However, a large proportion of the liposomal doxorubicin dose fails to work because it is sequestered within endolysosomal organelles following endocytosis of the liposomes due to the phenomenon of ion trapping. Listeriolysin O (LLO) is a pore-forming protein that can provide a mechanism for endosomal escape. The present study demonstrates that liposomal coencapsulation of doxorubicin with LLO enables a significantly larger percentage of the dose to colocalize with the nucleus compared to liposomes containing doxorubicin alone. The change in intracellular distribution resulted in a significantly more potent formulation of liposomal doxorubicin as demonstrated in both the ovarian carcinoma cell line A2780 and its doxorubicin-resistant derivative A2780ADR.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukaemia (CLL). The importance of this signalling pathway identifies it as a prime therapeutic target; however, the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. This article discusses the role of NF-κB in CLL as a common crossroad for pathways promoting drug resistance in CLL. We provide the background on how this pathway contributes to both spontaneous and drug-induced apoptosis. Potential new avenues to regulate this pathway in CLL are also discussed.

The rise of antibiotic resistance of common human pathogens and the lack of development of novel therapeutic treatments has created a threat to global health. A unique source for potential novel treatments are from microorganisms, particularly within the complex, antagonistic polymicrobial interactions that take place in microbial communities. These unique mechanisms utilized by microorganisms to fight each other could potentially identify novel therapeutic targets for use at a clinical level, however, there is a lack of research in this area to determine its applicability. Alcaligenes faecalis is a Gram-negative bacterium that seldom causes human disease and has been observed in our lab to show competitive, contact-dependent inhibitory mechanisms against Bacillus species, Candida albicans, and Staphylococcus species. These bacterial and eukaryotic microbes are increasingly a common source of human disease and all exhibit increased incidences of drug resistance. In this study, genetic elements related to A. faecalis’ contact-dependent inhibitory mechanism were determined via transposon mutagenesis. Genomic sequencing was performed on mutant strains of A. faecalis that exhibited diminished inhibition or loss-of-function inhibition of the competing microbes. In A. faecalis mutant strains P2-9 and P1-42, the interrupted gene was identified as a FAD-binding oxidoreductase with a 94% and 90% match of nucleotide sequence. Mutant strain P2-25’s interrupted gene was identified as an MFS transporter with a 100% match and P2-30’s interrupted gene was identified as a mechanosensitive ion channel with a 100% match. Further analysis of these mutants is needed to determine their role in the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus, C. albicans, and Bacillus treatments.

No description available.

Studies of drug resistance mostly characterize genetic mutations, and we know much less about the phenotypic mechanisms of drug resistance, especially at a quantitative level. p53 is an important mediator that regulates the cellular response to chemotherapy, but even cancer cells with wild-type p53 exhibited variable drug sensitivity for unclear reasons. In this PhD thesis, I investigated the mechanistic basis underlying differential p53 pathway activation in response to two types of chemotherapeutics, i.e., etoposide (a DNA-damaging drug) and 5-fluorouracil (5-FU, an antimetabolites), which led to distinct cell fate outcome in drug sensitive vs. resistant cancer cells. Specifically, I uncovered a new resistance mechanism to etoposide through bimodal modulation of p53 activation dynamics and characterized a four-component regulatory module, involving ATM, p53, Mdm2 and Wip1, which generates bimodal p53 dynamics through coupled feed-forward and feedback loops. Moreover, I found that the inhibitory strength between ATM and Mdm2 determined the differential modular output between drug sensitive and resistant cancer cell lines, and that combinatorial inhibition of Mdm2 and Wip1 was an effective strategy to alter p53 dynamics in resistant cancer cells and sensitize their apoptotic response, pointing to p53 pulsing as a potentially druggable mechanism that mediates resistance to DNA damaging chemotherapy. As for response to 5-FU, preliminary results illustrated that 5-FU activated p53 and differential cell fate outcome via ribosomal stress, rather than DNA damage. Different from dose response to etoposide, 5-FU-induced p53 activity was not only regulated by p53 induction level but also p53 phosphorylation by kinases, such as DNA-PK. Overall, this thesis presented original results that elucidated phenotypic mechanism of chemoresistance and provide new angles towards developing more effective combinatorial anticancer therapy.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).