Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
Browse
2 results
Search Results
Item CONTEXTUALIZATION OF THE E. COLI LSR SYSTEM: RELATIVE ORTHOLOGY, RELATIVE QS ACTIVITY, AND EMERGENT BEHAVIOR(2015) Quan, David Nathan; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Within bacterial consortia there exist innumerable combinatorial circumstances, some of which may tip the scale toward pathogenicity, some of which may favor asymptomatic phenotypes. Indeed, the lines and intersections between commensal, pathogenic, and opportunistic bacteria are not always clean. As a foothold to mediate pathogenicity arising from consortia, many have puzzled at communication between bacteria. Primary among such considerations is quorum sensing (QS). Analogous to autocrine signaling in multicellular organisms, QS is a self-signaling process involving small molecules. Generally, QS activation is believed to have pleiotropic effects, and has been associated with numerous pathogenic phenotypes. The research herein focuses on autoinducer-2 (AI-2) based QS signaling transduced through the Lsr system. Produced by over 80 species of bacteria, AI-2 is believed to be an interspecies signaling molecule. Outside of the marine bacteria genera Vibrio and Marinomonas, the only known AI-2 based QS transduction pathway is the Lsr system. We sought to deepen the characterization of the Lsr system in contexts outside of the batch cultures in which it was originally defined. First, we interrogated E. coli K-12 W3110 Lsr system orthologs relative to the same strain's lac system. Both systems are induced by the molecule which they import and catabolize. We searched for homologs by focusing on the gene order along a genome, as gene arrangement can bear signaling consequences for autoregulatory circuits. We found that the Lsr system signal was phylogenetically dispersed if not particularly deep, especially outside of Enterobacteriales and Pasteurellaceaes, indicating that the system has generally been conferred horizontally. This contrasts with the lac system, whose signal is strong but limited to a select group of highly related enterobacteria. We then modeled the Lsr system with ODEs, revealing bimodality in silico, bolstering preliminary experimental evidence. This bifurcated expression was seen to depend upon nongenetic heterogeneity, which we modeled as a variation of a single compound parameter, basal, representing the basal rate of AI-2 flux into the cell through a low flux pathway. Moreover, in our finite difference-agent based models, bimodal expression could not arise from spatial stochasticity alone. This lies in contrast with the canonical LuxIR QS system, which employs an intercellular positive feedback loop to activate the entire population. We examined the consequences of this contrast, by modeling both systems under conditions of colony growth using finite difference-agent based methods. We additionally investigated the confluence of Lsr signaling with chemotactic sensitivity to AI-2, which has been demonstrated in E. coli. Finally, the consequences of bimodality in interspecies interactions were assessed by posing two populations containing different Lsr systems against each other. While few natural consortia consist of only two interacting bacteria, these studies indicate that AI-2 based Lsr signaling may mediate a multitude of transitional intraspecies and interspecies bacterial dynamics, the specifics of which will vary with the context and the homologs involved.Item Nucleic Acid Extraction and Detection Across Two-Dimensional Tissue Samples(2010) Armani, Michael Daniel; Shapiro, Benjamin; Smela, Elisabeth; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Visualizing genetic changes throughout tissues can explain basic biological functions and molecular pathways in disease. However, over 90% of mammalian messenger RNA (mRNA) is in low abundance (<15 copies per cell) making them hard to see with existing techniques, such as in-situ hybridization (ISH). In the example of diagnosing cancer, a disease caused by genetic mutations, only a few cancer-associated mRNAs can be visualized in the clinic due to the poor sensitivity of ISH. To improve the detection of low-abundance mRNA, many researchers combine the cells across a tissue sample by taking a scrape. Mixing cells provides only one data point and masks the inherent heterogeneity of tissues. To address these challenges, we invented a sensitive method for mapping nucleic acids across tissues called 2D-PCR. 2D-PCR transfers a tissue section into an array of wells, confining and separating the tissue into subregions. Chemical steps are then used to free nucleic acids from the tissues subregions. If the freed genetic material is mRNA, a purification step is also performed. One or more nucleic acids are then amplified using PCR and detected across the tissue to produce a map. As an initial proof of concept, a DNA map was made from a frozen tissue section using 2D-PCR at the resolution of 1.6 mm per well. The technique was improved to perform the more challenging task of mapping three mRNA molecules from a frozen tissue section. Because the majority of clinical tissues are stored using formalin fixation and not freezing, 2D-PCR was improved once more to detect up to 24 mRNAs from formalin-fixed tissue microarrays. This last approach was used to validate genetic profiles in human normal and tumor prostate samples faster than with existing techniques. In conclusion, 2D-PCR is a robust method for detecting genetic changes across tissues or from many tissue samples. 2D-PCR can be used today for studying differences in nucleic acids between tumor and normal specimens or differences in subregions of the brain.