Biological systems display remarkable complexity that is not properly accounted for in small, reductionistic models. Increasingly, big data approaches using genomics, proteomics, metabolomics etc. are being applied to predicting and modifying the emergent phenotypes produced by complex biological systems. In this research, several novel tools were developed to assist in the acquisition and analysis of biological big data for a variety of applications. In total, two entirely new tools were created and a third, relatively new method, was evaluated by applying it to questions of clinical importance. 1) To assist in the quantification of metabolites at the subcellular level, a strategy for localized in-vivo enzymatic assays was proposed. A proof of concept for this strategy was conducted in which the local availability of acetyl-CoA in the peroxisomes of yeast was quantified by the production of polyhydroxybutyrate (PHB) using three heterologous enzymes. The resulting assay demonstrated the differences in acetyl-CoA availability in the peroxisomes under various culture conditions and genetic alterations. 2) To assist in the design of genetically modified microbe strains that are stable over many generations, software was developed to automate the selection of gene knockouts that would result in coupling cellular growth with production of a desired chemical. This software, called OptQuick, provides advantages over contemporary software for the same purpose. OptQuick can run considerably faster and uses a free optimization solver, GLPK. Knockout strategies generated by OptQuick were compared to case studies of similar strategies produced by contemporary programs. In these comparisons, OptQuick found many of the same gene targets for knockout. 3) To provide an inexpensive and non-invasive alternative for bladder cancer screening, Raman-based urinalysis was performed on clinical urine samples using RametrixTM software. RametrixTM has been previously developed and employed to other urinalysis applications, but this study was the first instance of applying this new technology to bladder cancer screening. Using a pool of 17 bladder cancer positive urine samples and 39 clinical samples exhibiting a range of healthy or other genitourinary disease phenotypes, RametrixTM was able to detect bladder cancer with a sensitivity of 94% and a specificity of 54%. 4) Methods for urine sample preservation were tested with regard to their effect on subsequent analysis with RametrixTM. Specifically, sterile filtration was tested as a potential method for extending the duration at which samples may be kept at room temperature prior to Raman analysis. Sterile filtration was shown to alter the chemical profile initially, but did not prevent further shifts in chemical profile over time. In spite of this, both unfiltered and filtered urine samples alike could be used for screening for chronic kidney disease or bladder cancer even after being stored for 2 weeks at room temperature, making sterile filtration largely unnecessary. / Doctor of Philosophy / Biological systems display remarkable complexity that is not properly accounted for in conventional, reductionistic models. Thus, there is a growing trend in biological studies to use computational analysis on large databases of information such as genomes containing thousands of genes or chemical profiles containing thousands of metabolites in a single cell. In this research, several new tools were developed to assist with gathering and processing large biological datasets. In total, two entirely new tools were created and a third, relatively new method, was evaluated by applying it to questions of medical importance. The first two tools are for bioengineering applications. Bioengineers often want to understand the complex chemical network of a cell’s metabolism and, ultimately, alter that network so as to force the cell to make more of a desired chemical like a biofuel or medicine. The first tool discussed in this dissertation offers a way to measure the concentration of key chemicals within a cell. Unlike previous methods for measuring these concentrations, however, this method limits its search to a specific compartment within the cell, which is important to many bioengineering strategies. The second technology discussed in this paper uses computer simulations of the cells entire metabolism to determine what genetic alterations might lead to better produce a chemical of interest. The third tool involves analyzing the chemical makeup of urine samples to screen for diseases such as bladder cancer. Two studies were conducted with this third tool. The first study shows that Raman spectroscopy can distinguish between bladder cancer and related diseases. The second v study addresses whether sterilizing the urine samples through filtration is necessary to preserve the samples for analysis. It was found that filtration was neither beneficial nor necessary.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/102706 |
| Date | 20 September 2019 |
| Creators | Huttanus, Herbert M. |
| Contributors | Biological Systems Engineering, Senger, Ryan S., Zhang, Chenming, Robertson, John L., Bevan, David R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
Page generated in 0.003 seconds