▼ Chlamydia trachomatis is an obligate intracellular microbe that is responsible for trachoma and chlamydia in much of the developing world, but is difficult to manipulate due to its biphasic developmental cycle. The current protocol for transforming Chlamydia trachomatis is a very time consuming process, yielding limited numbers of successful transformants only after multiple passages of infection which can take up to 48 hours per passage, while also requiring a highly time consuming purification process for elementary bodies. Chlamydia trachomatis’ genetically conserved and faster growing cousin, Chlamydia muridarum (MoPn) was used as a model organism to test the influence of elementary body (EB) purity and concentration of plasmid on transformation efficiency. Ultimately, the experimentation was inconclusive; higher amounts of plasmid in the initial transformation did not yield a higher number of transformants nor yield them in earlier generations when compared to the control groups. Furthermore, the purity of infectious elementary bodies (EBs) were not essential for transformational competence; both ultra-purified EBs that were isolated via a density gradient and partially purified Chlamydia muridarum displayed successful transformants. Further experimentation would be needed with higher sample sizes to ensure statistical significance, and trials on Chlamydia trachomatis itself would have to be performed to ensure consistency across both species. Finally, trimethoprim, banzal-N-acylhydrazones (BAH), and other antibiotics should continue to be screened as alternative selective agents due to their differences in mechanisms of action. Advisors/Committee Members: Runnels, Loren (chair), Fan, Huizhou (internal member), Fondell, Joseph (outside member), School of Graduate Studies.

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Alteration of glucose metabolism is a unique feature for a majority of cancers. Cancer cells exhibit aerobic glycolysis also known as the Warburg effect even in the presence of oxygen. During this mode of glucose metabolism, a majority of pyruvate is converted to lactate rather than entering mitochondria for complete oxidation through oxidative phosphorylation. The functional importance of aerobic glycolysis to cancer cells is becoming clear. Basically, aerobic glycolysis prevents pyruvate from complete oxidation inside mitochondria. This shunts glycolytic intermediates to pathways for synthesis of NADPH and building blocks of macromolecules, which are required for cell growth and proliferation. Pyruvate entrance into mitochondria is enhanced via mitochondrial uncoupling, a process that permits proton influx through the mitochondrial inner membrane without generating ATP. Consequently, mitochondrial uncoupling stimulates “idle ” oxidation of acetyl-CoA, leading to complete oxidation of glucose. Thus, we hypothesize that safe mitochondrial uncouplers could be strong anticancer agents to inhibit the anabolic role of the Warburg effect. We utilized two approaches to address this hypothesis. First, we tested two mitochondrial uncoupler compounds, NEN (niclosamide ethanolamine) and oxyclozanide, on their metabolic effects and anti-cancer activities. We used metabolomics NMR to study the effect of mitochondrial uncoupling on glucose metabolism in colon cancer MC38 cells. We further examined the anti-cancer effect of NEN and oxyclozanide in cell models and hepatic metastasis of colon cancer in animal model. We found that mitochondrial uncoupling stimulates pyruvate influx to mitochondria and decreases various anabolic pathway activities. Moreover, mitochondrial uncouplers arrest cell cycle progression, inhibit cell proliferation and reduce clonogenicity. Furthermore, oral treatment with mitochondrial uncouplers diminishes hepatic metastasis of colon cancer cells transplanted intrasplenically in mice. Second, we tested MB1-47, a novel mitochondrial uncoupler with good pharmacokinetic and toxicological profiles, in preventing and treating pancreatic cancer. Our study demonstrated that MB1-47 is effective in inducing mitochondrial uncoupling in pancreatic cancer cells and inhibits the proliferation of multiple murine and human pancreatic cancer cell lines. In the tumor xenograft mouse models, oral MB1-47 treatment exhibits excellent activity in preventing tumor growth and metastasis. Our data support a unique approach for targeting cancer cell metabolism for cancer prevention and treatment and identified prototype compounds for this mechanism.

Advisors/Committee Members: Fondell, Joseph (chair), Fan, Huizhou (internal member), Jin, Shengkan (internal member), Banerjee, Debabrata (outside member), School of Graduate Studies.

▼ Chlamydia trachomatis is a gram-negative bacterium and human pathogen responsible for the most prevalent sexually transmitted infection in the world. Epidemiologic studies indicate that the number of new C. trachomatis infections reported in the US likely approaches as much as 0.5 per a population of 100 every year. This, when paired with the fact that all current forms of treatment for C. trachomatis infections have unintended side effects including harm to gut and vaginal microbiota, has compelled many biologists to look to further research on C. trachomatis in hopes of developing new antichlamydial agents. In this thesis, I review several projects involving C. trachomatis that provide a framework for understanding both the growth and the developmental cycle of C. trachomatis. Through a mixture of in-depth proteomics, transcriptomics, and in vivo assays, I present five overall conclusions: (i) Lactobacilli-derived lactic acid, at concentrations that are physiological in the cervicovaginal lumen, can disrupt the outer membrane complex of and inactivate C. trachomatis; (ii) immunoprecipitation of chlamydial elementary bodies (EBs) using an antibody against a cell-surface protein of C. trachomatis is not an efficient method for purifying the bacterium; (iii) the growth of bacterial species that share significant sequence identity may not always be similar in media containing the same sera; (iv) the Chlamydia-specific transcription factor, GrgA, can stimulate transcription from promoters recognized by two different σ factors of C. trachomatis and has differential affinity for these two σ factors; (v) GrgA may associate with multiple subunits of the chlamydial RNA polymerase and, considering its role in transcription regulation and its specificity, may prove to be an excellent target for novel therapeutic agents against C. trachomatis. Advisors/Committee Members: Desai, Malhar, 1993- (author), Fondell, Joseph (chair), Fan, Huizhou (internal member), Millonig, James (internal member), Walworth, Nancy (internal member), School of Graduate Studies.

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Nuclease-dependent precise genome editing such as correction of point mutations requires introduction of targeted DNA double strand breaks (DSB) and activation of homology dependent repair (HDR), limiting its application to proliferating cells. To expand GE capabilities for therapeutic use in non-dividing somatic cells it is necessary to precisely modify nucleotides avoiding DSBs. Recently, Cas9-cytidine deaminase fusions, also known as based editors (BE), were shown to precisely modify target bases at certain genomic loci. To expand the base editing toolbox, we sought to engineer a novel base editing system based on RNA-aptamer mediated recruitment. To this end, we engineered a nuclease-deficient CRISPR/Cas9 system as a recruitment platform for non-nuclease DNA/RNA editing enzymes that catalyze C·G→T·A conversions by cytidine deamination. Targeted nucleotide modification was achieved with high precision in prokaryotic and eukaryotic cells. In bacteria, we tested our system targeting the rifampicin resistance determining region of the rpoB gene. Survival in rifampicin reached over 1000-fold higher than untreated cells. To examine whether the system can correct loss of function mutations in human genome, we treated a stably integrated non-fluorescent EGFP gene containing an A·T→G·C mutation on the chromophore sequence. Fluorescence was efficiently restored in treated cells, detecting around 10% of GFP positive cells after treatment. Next generation sequencing confirmed a G·C→A·T conversion in 60% of reads at the target position, restoring the wild type sequence, with low by-stander effect. Exome-wide sequence analysis revealed no detectable off-target effects. Targeting of endogenous loci also resulted in highly efficient nucleotide conversion at the desired C positions. We also show that our system can destroy the 3’ splice acceptor site of intron 50 in human DMD gene, potentially inducing exon 51 skipping, providing evidence of a therapeutic application to treat Duchenne muscular dystrophy. Taken together, the data show that our GE system represents a safe and promising technology for editing specific nucleotides, correcting genetic mutations or other clinically relevant applications, independent of DSB and HDR, with potential therapeutic value in non-dividing cells.

Advisors/Committee Members: Jin, Shengkan Victor (chair), Banerjee, Debabrata (internal member), Fondell, Joseph (internal member), Padgett, Richard (outside member), School of Graduate Studies.