1 / Yue Sun

Natural peptide products isolated from various organisms often contain N-methylated backbones. Such a modification of backbone of the peptide changes its conformational rigidity. This modification improves the biological properties of the peptide, such as improved target affinity, proteolytic stability or membrane permeability. Therefore synthesis of N-methylated peptide libraries is valuable in screening for drug-like peptides suitable for therapeutic uses. Protein synthesis using recombinant elements (PURE) and Flexizyme were used in order to reassign specific codons to N-methyl amino acids. mRNA-dependent translation system enable us to make our desired peptides with N-methyl amino acids. This technology is a convenient tool for the construction of N-methyl peptide libraries. Using Flexizyme in order to make library of N-methyl peptides requires significant amount of tRNA. Therefore developing a simple and rapid method for purification of specific tRNA from fully modified E. coli total tRNA would be advantageous Here we reported a new technique in purification of individual tRNAs using fluorous affinity tag. From total tRNA, desired tRNA could be charged with related amino acid and tagged with fluorous molecule through reductive amination.

Soft corals belonging to the genus Nephthea have been found to be a rich source of terpenoids and steroids. Chromatographic separation of acetone and CH2Cl2/MeOH extracts of Formosan soft coral Nephthea chabrolii resulted in the isolation of thirteen new steroids, including nine 4£\-methylated steroids (1−6, 8−10), three 19-oxygenated steroids (11−13), and a novel skeleton 19-norsteroid (7). The structures of these metabolites were elucidated by extensive spectral analyses and anti-inflammatory activities were measured in vitro. Compounds 1−2, 4−5, 7−11 and 13 at a concentration of 10 £gM significantly reduce the levels of iNOS (inducible nitric oxide synthase ). Compounds 2, 4, 6−7 and 10−11 at a concentration of 10 £gM also significantly reduce the levels of COX-2 (cyclooxygenase-2). Compounds 2, 4, 7, 10 and 11 can reduce the levels of both iNOS and COX-2. All compounds at a concentration of 10 £gM did not affect the housekeeping £]-actin protein expression.

Conversion of 1-Methylpentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹]undecane- 8,11-dione into the corresponding mono(ethylene ketal) followed by Wolff-Kishner reduction resulted in a mixture of two isomers (i.e., 1- and 7-methyl-8-[2',-(1',3',dioxolano)]pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹] undecane. Hydrolysis of each isomer in turn resulted in 1- and 7- methyl pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹ ]undecan-8-ones (i.e.,"methylated PCU-8-ones"), respectively. "Titanium-promoted reductive dimerization of each of the methylated pentacycloundecane (PCU)-8-ones afforded mixtures of "methylated PCU alkene dimers". Individual isomers have been isolated from these mixtures via column chromatography by using silver nitrate impregnated silica gel as adsorbent followed by fractional recrystallizations of individual chromatography fractions. Structures of three isomerically pure methylated PCU alkene dimers (C₂₄H₂₈) have been established unequivocally by application of single crystal X-ray crystallographic methods.

Role of N-methylated lysines (K) and arginines (R) was underappreciated for a long time before the turn of the century. With the help of new emerging technologies, their crucial role in chromatin regulation was established and now their mention when discussing gene regulation is almost a given. Despite this, much about how they contribute to the cellular chemistry is still to be discovered. There is a major gap in current knowledge base due to an incomplete list of possible lysine and arginine methylation sites. This is because of their low copy number inside the cell which makes it difficult to detect them. New methylation sites are being added every day. This thesis aims to provide a solution to this problem by establishing methods that can help detect N-methylated lysines and argnines that are present in really low quantity inside the cell. The work is influenced by a previously established fact that p-sulfonatocalix[4]arene binds methylated lysine over unmethylated ones. We have first attempted to improve this native affinity by decorating the calix[4]arene skeleton with different substituents. To this effect, we have developed methods for regioselective functionalization of calix[4]arene scaffold and then studied their effect on its binding profile against a set of test peptides derived from proteins found in vivo. We then demonstrate a proof-of-concept enrichment method using selected molecules from our inventory. We use these calix[4]arene based molecules as a stationary phase modifier in a chromatography setup and then show that it can separate peptides based on presence of N-methylated lysines and arginines. We propose that introduction of such a method would improve the visibility of low level N-methylated peptides by removing the bulk of back ground unmethylated analytes and thus improving their signal strength. Finally, we establish the utility of this method by showing that more N-methylated lysines are detected from a real-world proteomics sample prepared using our enrichment method. This work opens new avenues for use of supramolecular chemistry in proteomics studies. We believe that this thesis is a confident demonstration that host-guest chemistry can help expand the existing knowledge about bimolecular processes found in vivo and must be explored further. / Graduate / 2021-12-23

RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer. Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing a reassembly and activation of the enzyme. In the presence of a substrate, the activated β-lactamse enzyme hydrolyzes the β-lactam bond in the substrate and shows a color change from yellow to red in the presence of a methylated cognate DNA. The study suggests that a solution based assay was not as specific in differentiating signal intensities between methylated and non-methylated DNA. It was also not sensitive in measuring dose dependent signals. Zinc finger array could successfully show relatively low signals for non-methylated DNA. The findings of the study show that MBD2 shows higher preference for mCpG than MBD1 in the mCpG SEER-Lac system and oligonucleotides with a 2 bp spacing between methylation and ZF target site shows higher signals than the 3 bp spacing. Due to it’s specificity and sensitivity, it serves as a potential diagnostic tool to detect cancer.

Terpenes comprise a large diverse class of natural products and many of them attract interest because of their physiological function, therapeutic and industrial values. Triterpene oils including squalene (C30), botrycococcene (C30) and their methylated derivatives (C31-C37) generated by the green algae Botryococcus braunii race B, which have recently received significant attention because of their utility for advanced biofuels. However, the slow growth habit of B. braunii makes it impractical as a robust biofuel production system. In this thesis, we firstly evaluated the potential of generating high levels of triterpene (C30) production in tobacco plants by diverting carbon flux from cytosolic MVA pathway or plastidic MEP pathway by overexpressing avian farnesyl diphosphate synthase along with triterpene synthase targeted to the cytoplasm or the chloroplast of cells. Up to 1,000 µg/g fresh weight of squalene and 544 µg/g fresh weight of botryococcene was achieved in our transgenic plants with this metabolism direct to the chloroplasts, which is about approximately 100-times greater than that accumulating in the plants engineered for cytosolic production. To test if methylated triterpenes can be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) into wild type plants and transgenic tobacco plants selected for high level triterpene accumulation. We observed that up to 91% of the total triterpene content was converted to methylated forms (C31, C32) by targeting the TMTs to the chloroplasts of transgenic plants, whereas only 4-14% of total triterpenes were methylated when TMTs were directed to the cytoplasm. Select transgenic lines were growing in field studies from 2011 to 2014 to evaluate their physiological performance under field conditions. Surprisingly, the field studies suggested that the growth and agronomic performance of the transgenic lines accumulating squalene were not compromised, while those accumulating high levels of botryococcene were only 72%-76% as tall, had about 59%-75% of the leaf area, and about 55%-75% of the biomass as wild type plants. Yet, these transgenic plants had photosynthetic capacity equal to the wild type plants.

There is limited research on High Surfactant Oil Concentrates (HSOC), so studies were conducted for their evaluation. Multiple MSO-based (HSMOC) and POC-based (HSPOC) HSOCs were tested with glyphosate plus dicamba or glyphosate plus tembotrione. The addition of HSMOCs provided greater indicator species control HSPOCs when added to either herbicide tank-mix. When multiple experimental oil to surfactant ratios were added to glyphosate plus dicamba or glyphosate plus tembotrione, there were no differences among experimental HSOC ratios when added to either tank-mix by 28 days after application. Since oil adjuvant rates can be based on either treated area or percent of spray solution, oils were added to either dicamba or tembotrione to evaluate rate methods. There were few differences in species control when oils were added to dicamba. Quinoa and amaranth control were more consistent when using the percent volume-based rates with tembotrione.

DNA methylation is an epigenetic mechanism that usually occurs at CpG sites in the genome. Both sequencing and array-based techniques are available to detect methylation patterns. Whole-genome bisulfite sequencing is the most comprehensive but cost-prohibitive approach, and microarrays represent an affordable alternative approach. Array-based methods are generally cheaper but assess a specific number of genomic loci, such as Illumina methylation arrays. Differentially methylated regions (DMRs) are genomic regions with specific methylation patterns across multiple CpG sites that associate with a phenotype. Methylation at nearby sites tends to be correlated, therefore it may be more powerful to study sets of sites to detect methylation differences as well as reduce the multiple testing burden, compared to utilizing individual sites. Several statistical approaches exist for identifying DMRs, and a few prior publications compared the performance of several commonly used DMR methods. However, as far as we know, no comprehensive comparisons have been made based on genome-wide simulation studies. This dissertation provides some comprehensive suggestions for DMR analysis based on genome-wide evaluations of existing DMR tools and presents the development of a novel approach to increase the power to identify DMRs with clinical value in genomic research. The second chapter presents genome-wide null simulations to compare five commonly used array-based DMR methods (Bumphunter, comb-p, DMRcate, mCSEA and coMethDMR) and identifies coMethDMR as the only approach that consistently yields appropriate Type I error control. We suggest that a genome-wide evaluation of false positive (FP) rates is critical for DMR methods. The third chapter develops a novel Principal Component Analysis based DMR method (denoted as DMRPC), which demonstrates its ability to identify DMRs using genome-wide methylation arrays with well-controlled FP rates at the level of 0.05. Compared to coMethDMR, DMRPC is a robust and powerful novel DMR tool that can examine more genomic regions and extract signals from low-correlation regions. The fourth chapter applies the new DMR approach DMRPC in two “real-world” datasets and identifies novel DMRs that are associated with several inflammatory markers.

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