Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 78-94).

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Molecular docking technologies enable the extraordinary structural diversity of natural products to be harnessed in an efficient manner. In this thesis, in silico techniques were used to discover and develop c-myc oncogene down-regulators and JAK2 inhibitors from databases of natural products and approved drugs. In Chapter 1, current literature on the use of molecular docking in virtual screening for the identification of bioactive molecules from natural product databases are reviewed. Chapter 2 provides an overview of the experiments performed during the course of this work, including molecular docking, PCR stop assay, absorption spectroscopy, CD spectroscopy, FID assay, mass spectrometry, SPR spectroscopy, ELISA assay, MTT assay, luciferase assay and Western blot analysis. The mechanisms, applications and protocols of these experiments are detailed. A unique intramolecular G-quadruplex c-myc NHE IIIi loop isomer model developed by our group was employed to design and screen 30 flavone derivatives in silico. The highest-scoring flavone derivatives 3.5, 3.6 and 3.7 containing cationic pyridinium side chains that could interact with the G-quadruplex grooves were synthesized. The flavone derivatives could stabilize the c-myc G-quadruplex in the PCR-stop assay, and induce the G-quadruplex structure in guanine-rich sequences as revealed by CD spectroscopy. The binding affinity of the derivatives towards various DNA structures was examined using UV-visible spectroscopy. The most promising derivative 3.7 was further subjected to surface plasmon resonance spectroscopy, in silico molecular modeling and luciferase reporter assay to determine its selectivity, binding interaction mode and c-myc G-quadruplex promoter inhibitory activity in cancer cells. This compound also displayed promising cytotoxic behavior against human cancer cell lines. This part of work is detailed in chapter 3. Chapter 4 describes the application of computer-aided techniques for the repurposing of FDA-approved drugs as c-myc oncogene G-quadruplex stabilizers. Methylene blue (MB) emerged as a promising scaffold after virtual screening of 3,000 FDA-approved drugs. A structure-based lead optimization approach was used to generate and screen 50 MB derivatives, containing side chains that could interact with the G-quadruplex grooves, in silico. The highest-scoring compounds 4.10, 4.11 and 4.12 were synthesized and their ability to interact with the c-myc G-quadruplex was investigated using FID assay. The most promising compound 4.11 stabilized c-myc G-quadruplex DNA in a PCR-stop assay. The selectivity of 4.11 for the c-myc G-quadruplex over duplex DNA and other G-quadruplexes was demonstrated using UV-visible spectroscopy and mass spectrometry. Compound 4.11 could induce or stabilize c-myc G-quadruplex formation in cellular models, and displayed higher cytotoxicity against human hepatocarcinoma cells compared to the parent compound, MB. The application of the DOLPHIN kinase model to discover natural product scaffolds as Type II JAK2 inhibitors is presented in chapter 5. Amentoflavone, a biflavonoid from the Chinese plant Gingko biloba, emerged as a promising candidate after biological verification of the hit structures. Amentoflavone was optimized in silico, and the top scoring derivatives were synthesized. The activity of the amentoflavone analogues against JAK2 activity in HEL cells was evaluated using a Western blot assay. Two derivatives, 5.3 and 5.7, showed low-micromolar activity against JAK2 phosphorylation in cellulo. Analogue 5.2 inhibited total JAK2 content in HEL cells and also displayed potent anti-proliferative activity against HEL cells in the MTT assay. This chapter also describes the total synthesis of amentoflavone.

Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.

It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).

The Caenorhabditis elegans somatic gonad is patterned by the activity of regulatory cell types, which govern its morphology, serve as the germline niche, and pattern its connection to the outside. All regulatory cell types are specified by activity of the basic helix-loop-helix gene hlh-2/E/Daughterless, and differences in how functions are assigned between the regulatory cells in males and hermaphrodites lead directly to their sexually-dimorphic gonads. Here, I present evidence that a code of bHLH genes function together with hlh-2 to promote the specification and function of each regulatory cell type except for the hermaphrodite anchor cell, which is specified by HLH-2 activity alone. Each regulatory cell type expresses an overlapping but distinct set of bHLH genes, which we find are required for its specification and associated functions. Notably, ectopic expression of regulatory cell bHLH complements are sufficient to transform cells with anchor cell potential into the expected regulatory cell, albeit transiently, suggesting that they are master regulators of regulatory cell fate. As all nematode species pattern their gonads through cognate regulatory cells and bHLH genes are highly conserved, we hypothesized that a similar bHLH code might function in specifying the regulatory cells of other species. In some nematode species the anchor cell, which remains stationary in C. elegans, is able to migrate. In C. elegans, the bHLH gene hlh-12 is necessary for proper migration of hermaphrodite distal tip cells and male linker cell, the two migrating regulatory cell types; addition of hlh-12 to the C. elegans anchor cell causes it to become displaced in a manner dependent on the endogenous hermaphrodite distal tip cell and male linker cell machinery, suggesting that the anchor cell gains the ability to migrate with the addition of hlh-12. We thus hypothesized that ectopic expression of an hlh-12 ortholog in these species might have led them to evolve migrating anchor cells. However, phylogenetic analysis of the bHLH genes of several other species, including the ones with migratory anchor cells, suggests that hlh-12 may be novel to the Caenorhabditis genus and does not have orthologs in the species with migrating anchor cells, raising the possibility that either these species use another bHLH gene for migration or that their regulatory cells are specified in a bHLH-independent manner.

Ferroptosis is a form of regulated cell death that results in an accumulation of toxic lipid hydroperoxides. It has been implicated in several human disease models, including cancers, organ failure, and neurodegeneration. Identifying novel regulators and biomarkers for ferroptosis can result in identification of disease states where treatment with ferroptotic inducers or inhibitors can result in a positive outcome for a patient. In this thesis, I will describe several methods that were used to identify novel genetic regulators of ferroptosis. A CRISPR-Cas9-mediated, whole-genome loss of function screen identified several gene knockouts that resulted in a protective phenotype against ferroptosis. These genes were found to play a role in antioxidant response, glutathione bioavailability, and/or labile iron homeostasis consistent with the current model of ferroptotic cell death. Alternatively, a CRISPR-dCas9-mediated whole genome activation screen found Gch1 overexpression to have a potent anti-ferroptotic effect. Gch1 overexpression resulted in an increase to the basal levels of endogenous antioxidants including BH₄ and CoQ₁₀, as well as protection of a specific class of phospholipids containing polyunsaturated fatty acyl (PUFA) chains. This multipronged defense resulted in strong suppression of ferroptosis. Additionally, we were interested in the protection of this unusual class of phospholipids with two PUFA tails, and if their enrichment would result in a change in sensitivity to ferroptosis. We found that the degrees of unsaturation of the acyl chains, as well as differences in head groups of the phospholipids resulted in significant differences in the ability of the phospholipids to induce a strong ferroptotic response. The study of their uptake, subcellular localization, and remodeling pathways can further elucidate biological pathways of ferroptosis.

Sessile bacterial communities which form on the solid surface or solid-liquid interface are known as biofilms. Both single species and multispecies biofilms are characterized by an extracellular matrix of polymeric substances which gives them several hundred times more antibiotic resistances than a planktonic bacterial culture. Though bacteria are the most common causative agent of various diseases, because of the high antibiotic resistance, biofilms cause complications of various diseases like cystic fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated urinary tract infections and several other diseases. From past studies, quorum sensing has been established as a novel target mechanism against biofilms; in this study, the two-component signal transduction systems (2CSTSs) have been focused. Once better understood, 2CSTSs can serve as a novel drug target and prevention mechanism for biofilm associated diseases. According to prior high-throughput experiments and phenotype microarray experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the global regulator FlhD/FlhC were hypothesized to have an important effect on various developmental stages of biofilm formation. From that past study, we postulated that acetate metabolism may be an important aspect for biofilm formation. In this study, we tested and confirmed this hypothesis. We observed biofilms formed by several mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of the acetate mutants when compared to their isogenic parental Escherichia coli strain. An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms whereas an additional mutation in dcuR results in the formation of less biofilms. So the structural and the quantitative differences of acetate mutant biofilms depend on additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The future aim of this study is to study the temporal as well as the spatial gene expression of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter plate assay was performed to study the temporal expression from the promoters by quantifying the fluorescence intensity in the planktonic culture. According to this experiment, the highest expression of flhD was after 20 hours whereas, the expression of ompR increases up to 7 days, which indicates that the flhD expresses earlier than ompR. The decreasing phase of flhD expression was paralleled by the sharpest increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD expression. / National Institutes of Health (NIH grant 1R15AI089403) / United States. Animal and Plant Health Inspection Service

Mouse epiblast stem cells (EpiSC) are pluripotent cells derived of the epiblast of post-implantation blastocysts that can self-renew indefinitely in culture, display lineage-restricted differentiation, and appear to closely resemble human embryonic stem cells (ESC). Despite significant advances in the last decade, the precise molecular mechanisms and many master regulator (MR) genes underlying stem cell self-renewal, pluripotency, interactions with surrounding cells, and lineage-specific differentiation still remain elusive. The goal of this thesis is to address these gaps of knowledge using a systematic approach to identify novel MR genes and functionally validate them using genetically modified mouse models.In order to elucidate MR genes that control understudied biological processes, previous work in the Shen lab have computationally reconstructed the regulatory network of EpiSC and interrogated the EpiSC interactome with pluripotency signatures of EpiSC lines. One MR gene of interest from the previous analysis is ZC3H13, which encodes a protein that has been previously shown to be a crucial for N6-methyladenosine modification in RNA (m⁶A). This suggests a novel connection between m⁶A epitranscriptional modifications and primed state pluripotency. In my thesis research, I have shown that Zc3h13 is essential for proper trophoblast lineage differentiation and the importance of m6A modifications in early embryonic development. Using two Zc3h13 knockout mouse lines, I have found that Zc3h13 null embryos are embryonic lethal at the peri-implantation stage due to a failure to implant into the uterus. In vitro outgrowth analysis revealed a lack of trophoblast giant cells in Zc3h13 null outgrowths, and the lack of enlarged nuclei in the Zc3h13 null outgrowth suggests a failure in endoreduplication. Immunofluorescence analysis of Zc3h13 null blastocysts showed that the trophectoderm cells of Zc3h13 null blastocyst expressed trophectoderm specific factors at abnormal levels, indicating a severe dysregulation of the trophectoderm regulatory network. To elucidate the effects of Zc3h13 knockout on pluripotency, I also performed a detailed immunofluorescence analysis of Zc3h13 null inner cell mass (ICM), which expressed pluripotency factors at normal levels. However, Zc3h13 null blastocysts were less efficient at generating ESC lines and the Zc3h13 KO ESC generated were morphologically abnormal. Dot blot and mass spectrometry analysis showed that Zc3h13 KO ESC had a dramatically lower level of m⁶A modification, suggesting a connection between m6A epitranscriptional modification and endoreduplication. Interestingly, chimera and teratoma analysis showed that while Zc3h13 KO ESC can contribute to derivatives of the three primary lineages, Zc3h13 KO ESC has a bias towards neuroectoderm differentiation. In this thesis, I have shown the importance of m6A transcriptional regulation in trophoblast giant cell differentiation. Taken together, my studies can help further the understanding of the biological functions of m⁶A modifications as well as the relationship between transcriptional regulation and cell fate transition. My work highlights another level of gene regulation through epitranscriptional modification and the importance of the epitranscriptomic landscape in cell fate transition and development.