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Susceptibility of 112 strains of Staphylococcus aureus obtained from Dameron Hospital, Stockton, California was tested with 18 antimicrobials . The MIC method was used with the following antimicrobials : tetracycline, oxacillin, penicillin, ampicillin, vancomycin, cefazolin, erythromycin, clindamycin, gentamycin, rifampin, trimethoprimsulfamethoxazole, chloramphenicol, and cefotaxime . The standard Kirby-Bauer disc diffusion method was used to test neomycin, tobramycin, and amikacin . Methicillin, oxacillin, and nafcillin were tested with a modified Kirby-Bauer method, which included the addition of a 4% salt supplement to the media, incubation at 32C, and readings at both 24 and 48 hours. Comparing results of this study with those of Hall (1975), suggested that resistance to the following antibiotics has increased: penicillin, ampicillin, erythromycin, neomycin, gentamycin, methicillin, oxacillin, nafcillin, cefazolin, and clindamycin . Resistance to tetracycline has decreased. No resistance to chloramphenicol or vancomycin was encountered in either study . Of the 112 strains studied, 13 . 4% were susceptible to all antibiotics tested. Twelve patterns of resistance were identified : 0 . 9% were resistant to neomycin only, 1.8% to erythromycin only, 63.9% to both penicillin and ampicillin, and 20 . 0% were multiply- resistant . Nine patterns of multiple-resistance were found, involving a minimum of three antibiotics and a maximum of nine . Three MRSA strains were identified from out-patient isolates; no in-patient isolates were methicillin-resistant . The study suggests that MRSA strains are not a problem at Dameron Hospital, but identification of this group would be more accurate if incubation of the MIC panels is maintained for at least 24 hours at ~35C . It was found that the MIC method of antimicrobial susceptibility testing is more reliable than the Kirby-Bauer disc diffusion method for detection of methicillin-resistance. Problems involved in identification of heteroresistant staphylococci are discussed .

Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.

Malaria affects hundreds of millions of people worldwide and equally claims hundreds of thousands of lives each year. With the current spread of drug resistance to standard antimalarial drugs like chloroquine and the emergence of artemisinin-resistant parasites, new antimalarial drugs and formulations are urgently needed. An ethnobotanical survey was carried out in this study in search of novel compounds with promising antiplasmodial activity. Using the ethnobotanical approach, a total of 61 plant species from 59 genera distributed in 34 plant families were found to be used traditionally for the treatment of malaria in Nigeria. Biological evaluation of the plant¿s methanolic extracts was assessed using the parasite lactate dehydrogenase (pLDH) assay against the chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. A total of five (5) plant species showed more potent antiplasmodial activities against the malaria parasites. These are Acanthospermum hispidum, Cassia occidentalis, Kaempferia aethiopica Prosopis africana and Physalis angulata with MIC values ranging between 7.815µg/ml to 31.25µg/ml (3D7 strain) and 15.63µg/ml to 62.50µg/ml (K1 strain) against the malaria parasites, respectively. Two plants, Prosopis africana (Leguminosae-mimosoideae) and Physalis angulata (Solanaceae) were selected for further study. The phytochemical investigation of the active chloroform extracts of P. africana and P. angulata yielded several compounds with three known alkaloids, namely, prosopinine (I), prosopine (II) and acetamide (III). Their structures were confirmed by MS, 1D and 2D NMR spectroscopy. Compounds I, II and III have moderate in vitro antiplasmodial activity against the malaria parasites. Both chloroquine and artemether were used as standard control. / Association of Commonwealth Universities and the Commonwealth Scholarship Commission in the UK (Commonwealth Scholarship Reference Number: NGCS-2005-259).

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key A- and C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature’s biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Chapter 1 provides background on antibiotics, and the tetracycline class in particular, and the metabolic engineering and directed evolution techniques available to us for heterologous expression of enzymes in yeasts. In Chapter 2, we describe the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts. Further, in Chapter 3, we highlight our Tang Laboratory collaborators’ work, where they reported the heterologous biosynthesis of a non-antibiotic fungal anhydrotetracycline derivative, TAN-1612, in S. cerevisiae from Aspergillus niger. We have built upon this system, allowing for the high-titer production of TAN-1612 in yeasts. Finally, in Chapter 4, we outline our efforts to convert TAN-1612 into a high titer tetracycline- and analog-producer by modifying the 2-, 4-, and 6-positions, proven critical for antibiotic activity. By hijacking biosynthetic hydroxylating and reducing enzymes, we attempted to modify the 6α-position, dearomatizing the C-ring. We also expressed heterologous enzymes within the TAN-1612 pathway that could furnish the 2-position with a carboxamido group instead of its natural hydrogen groups. By taking advantage of yeast’s natural biosynthetic pathways, we will create inexpensive, single-dose antibiotics, setting the stage to pursue yeast as a novel therapeutic. These state-of-the-art synthetic biology technologies will create entirely new paradigms, leading the charge against infections and diseases.

Despite rapid advances in clinical oncology, acquired drug resistance still poses a significant threat to the long-term efficacy of current treatment regimens. Because most chemotherapy drugs aim to activate apoptosis in cancer cells, expansion of the pharmacopeia to include treatments targeting novel tumor cell death mechanisms is a promising anti-cancer strategy. Induction of ferroptosis, an iron-dependent form of regulated cell death, shows particular therapeutic potential as aggressive metastatic and drug-resistant cancer cell states have been demonstrated to possess an exquisite dependency on glutathione peroxidase 4 (GPX4), a key suppressor of the ferroptotic cell death pathway. However, current GPX4 inhibitors are limited by poor pharmacokinetic properties that preclude their clinical use. The development of novel drug-like GPX4 inhibitors would benefit from the discovery of new chemical scaffolds to both enhance our understanding of the structural basis of small molecule binding and inhibition as well as facilitate the rational design of future GPX4-targeted therapeutics. In this dissertation, we employed three high-throughput screening strategies to identify novel scaffolds of interest for GPX4 inhibitor development. First, a Lead-Optimized Compound (LOC) library was screened and we conducted further characterization and structure-activity relationship (SAR) studies on hit compound LOC880. Compared to the original hit, analogs QW-095 and QW-105 showed improved binding affinity and GPX4 inhibitory activity in vitro and also induced lipid peroxidation in cells suggestive of ferroptotic death. Further enhancement of the potency and ferroptosis specificity of this scaffold is still needed, but the potentially noncovalent and allosteric mechanism of action presents a novel approach for targeting GPX4. Second, we conducted extensive SAR analyses on another promising hit from the LOC library screen, LOC1886, which led to the identification of the lead compound QW-314. This analog showed significantly improved potency and ferroptosis specificity in multiple cancer cell contexts, including a drug-tolerant persister cell model of minimal residual disease. Characteristic markers of GPX4 inhibition and ferroptosis are also observed in cells treated with QW-314, including GPX4 protein degradation and induction of lipid peroxidation, and QW-314 exhibited excellent selectivity for GPX4 over another glutathione peroxidase family selenoprotein GPX1 in an in vitro assay using cell lysates. Moreover, we determined a baseline of pharmacokinetic measures including aqueous solubility and metabolic stability in human and mouse liver microsomes for further medicinal chemistry optimization. Lastly, we screened a DNA-encoded library (DEL) and an Enamine Diversity library, identifying 10 additional chemical starting points for future investigation.

The repeated emergence of Plasmodium falciparum resistance to first-line antimalarial drugs necessitates understanding the underlying resistance mechanisms to detect and monitor resistance in the field and to inform drug discovery efforts. With the advent of the FRG NOD human liver-chimeric (huHep) mouse model for P. falciparum genetic crosses, interest has renewed in harnessing this forward genetics tool to study traits including drug resistance. The antimalarial quinine (QN) is of particular interest as it has retained efficacy over 400 years as parasite resistance has been slow to develop against the drug, likely due to a multifactorial mechanism of which only several genes have been partially implicated. Chloroquine (CQ) is a former first-line drug for P. falciparum (that is still in use for P. vivax), and it’s phasing out has been associated with the recent emergence of CQ-sensitive P. falciparum parasites. While the CQ resistance transporter (PfCRT) is known to be the primary driver of resistance, studies have provided evidence for secondary modulators of CQ, of which only the multidrug resistance protein 1 (PfMDR1) transporter has been identified. This thesis addresses the hypotheses that additional mediators are involved in the parasite resistance mechanism to QN and that genes other than pfmdr1 modulate parasite resistance to CQ. In chapter 3, we present the P. falciparum genetic cross that we conducted between the QN- and CQ-sensitive African NF54 and QN- and CQ-resistant Cambodian Cam3.II parasites in huHep mice, in collaboration with Dr. Photini Sinnis’s laboratory at Johns Hopkins University. By applying different selective conditions to cross progeny bulk pools prior to cloning these bulks, we were able to recover 120 unique recombinant progeny from this cross. We observed minimal overlap in the progeny genotypes obtained from CQ and QN pressure, suggesting distinct mechanisms for parasite resistance to these drugs. Bulk progeny selection and progeny clone-based QN linkage mapping approaches identified quantitative trait loci (QTLs) on chromosomes 7 and 12, as well as minor QTLs on other chromosomes, consistent with a multifactorial resistance mechanism. We applied the latter approach to investigate parasite response to CQ and its active metabolite monodesethyl-CQ (md-CQ) and identified a novel chromosome 12 QTL in addition to pfcrt. Interestingly, while the chromosome 12 QTLs overlapped, the chromosome 7 QTL for high-grade QN resistance did not contain pfcrt. In chapter 4, we used bioinformatic approaches, whole-genome sequence data from our cross and field isolates, and literature review to identify the drug/metabolite transporter 1 (DMT1) as the top candidate of the chromosome 7 QTL, and S-adenosylmethionine mitochondrial carrier protein (SAMC), hydroxyethylthiazole kinase (ThzK), and ATP-dependent zinc metalloprotease (FtsH1) as the top candidates for the chromosome 12 QTLs. By harnessing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing methodologies (SNP-editing, knockout, and tagging), we obtained evidence favoring DMT1 as a marker of QN resistance and localized this transporter to structures associated with vesicular trafficking, PVM, lipid bodies, and the lysosome-like digestive vacuole. We also harnessed SNP-editing and identified FtsH1 as a potential mediator of QN resistance and a modulator of CQ and md-CQ resistance. QN, mefloquine, and lumefantrine belong to the same aryl-amino alcohol class, and we found that QN is structurally more similar to mefloquine than lumefantrine. We also showed that QN can partially inhibit heme detoxification. While conducting the work outlined in chapters 3 and 4, we identified an unmet need for quickly identifying clonal recombinant progeny and validating parasite identity, which inspired the study presented in chapter 5. We developed a genotyping method that can assess drug resistance-conferring SNPs directly from P. falciparum culture or infected blood as well as a multiplexed microsatellite genotyping method with five broadly informative markers. Both methods were applied in chapter 3 to identify clonal recombinant progeny, and the SNP genotyping method was used in chapter 4 to validate gene editing and progeny identity. We also tested the resolution, sensitivity, time, and cost of each method as well as whole-genome sequencing and recommended the ideal application for each genotyping method. Our data demonstrate that DMT1 is a novel marker for QN resistance, and a new chromosome 12 locus associates with CQ response, of which ftsh1 is a potential candidate. In chapter 6, we discuss the potential mechanisms by which DMT1 is involved in QN resistance, the potential impact of our findings, and future experiments that can further characterize the QN and CQ resistance mechanisms and the functional role of these candidate genes.

The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world’s population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient’s sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.

Due to the misuse of antibiotics, multi-drug resistant (MDR) bacteria have become more rampant in our society; these MDR have given rise to diseases that are not readily curable. One such agent is the Mycobacterium tuberculosis complex, which is a causative agent of tuberculosis (TB). Timely diagnostics of the bacterial infection and detection of bacterial drug-susceptibility profiles helps to initiate the necessary treatment in a timely fashion and to limit transmission of the disease. For more affordable detection of bacterial diseases, such as TN, tag-free split aptamer probes are advantageous. This proposal aims at designing split aptamer probes for detection of point mutations in the rpoB and katG genes of M. tuberculosis that are associated with resistance to two front-line antibiotics – rifampin and isoniazid, respectively, which causes MDR-TB. The probes will be designed and tested with synthetic oligonucleotide mimics of the bacterial genes in terms of their limit of detection and selectivity in discriminating the targets with single-nucleotide substitutions.

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