Chapter Two Abstract
The environmental impacts of hydraulic fracturing, particularly those of surface spills in aquatic ecosystems, are not fully understood. The goals of this study were to: (1) understand the effect of previous exposure to hydraulic fracturing fluids on aquatic microbial community structure and (2) examine the impacts exposure has on biodegradation potential of the biocide glutaraldehyde. Microcosms were constructed from hydraulic fracturing-impacted and non-hydraulic fracturing-impacted stream water within the Marcellus shale region in Pennsylvania. Microcosms were amended with glutaraldehyde and incubated aerobically for 56 days. Microbial community adaptation to glutaraldehyde was monitored using 16S rRNA gene amplicon sequencing and quantification by qPCR. Abiotic and biotic glutaraldehyde degradation was measured using ultra performance liquid chromatography—mass spectrometry and total organic carbon. It was found that non-hydraulic fracturing-impacted microcosms biodegraded glutaraldehyde faster than the hydraulic fracturing-impacted microcosms, showing a decrease in degradation potential after exposure to hydraulic fracturing activity. Hydraulic fracturing impacted microcosms showed higher richness after glutaraldehyde exposure compared to non-impacted streams, indicating an increased tolerance to glutaraldehyde in hydraulic fracturing impacted streams. Beta diversity and differential abundance analysis of sequence count data showed different bacterial enrichment for hydraulic fracturing-impacted and non-hydraulic fracturing-impacted microcosms after glutaraldehyde addition. These findings demonstrated a lasting effect on microbial community structure and glutaraldehyde degradation potential in streams impacted by hydraulic fracturing operations.
Chapter Three Abstract
Adaptive thermogenesis has re-emerged as an important component of energy expenditure. N-acyl amino acids (N-AAAs) can contribute to adaptive thermogenesis by uncoupling mitochondrial respiration. Hypothesizing that lipolysis promotes the formation of N-AAAs, the connection was studied in an avian model (chickens) because lipolysis is readily induced by acute (~ 4 hr) food withdrawal. An avian model also allowed evaluation of N-AAA production in the absence of brown adipose tissue, which was reported to contribute the enzyme (peptidase M20 domain containing 1 (PM20D1)) that catalyzes their synthesis. Serum samples were collected from 21 d broiler chicks that were fed ad libitum, fasted for 4, 8, or 12 h, or fasted for 12 h and then re-fed for 2 h (n=10/group). Samples were profiled for levels of 27 N-AAA species using a novel UPLC-HRMS method. In short, a single Ultra-Performance Liquid Chromatography—High Resolution Mass Spectrometry (UPLC-HRMS) method was developed and validated with 27 N-acyl amino acid standards and 20 bile acid standards. (Given their shared relevance for obesity research and their similar physio-chemical properties, a single analytical method for the simultaneous detection of these metabolites is desirable). Reversed-phase chromatography and Orbitrap mass spectrometry were used in tandem to detect the 47 metabolites in a 20.5-minute method. The standards were detected with a high degree of mass accuracy (less than 2 ppm for all N-AAAs and less than 7 ppm for all bile acids). In addition, protocols for the extraction of these metabolites from biological media were tested. Fasting progressively increased the serum pool of N-AAAs (p=1.8-06), in parallel with increased non-esterified fatty acid (NEFA) levels (p=0.0003). Refeeding for 2 h rapidly restored the N-AAA pool to fed levels (p=0.99; fed vs. 12 h fast-refed). Percent abundance of some species (e.g., oleoyl-l-valine; p=2.8-04) increased with fasting while others (e.g., stearoyl-l-leucine; p=1.9-05) declined, suggesting molecule-specific regulation of N-AAA synthesis. Expression of PM20D1 in adipose tissue (subcutaneous and abdominal) and in liver was not affected by fasting. Effect of diet on N-AAA expression was also evaluated using the same boiler chick model. Four unique diets, containing a supplement of flaxseed, canola, fish, or animal (lard) oil were administered to chicks, which were then sacrificed for analysis of N-AAA in tissue. The fatty acid composition of each diet was 42 directly reflected in the tail composition of each N-AAA profile. This suggests that substrate availability may be a prominent means of N-AAA regulation.
Chapter Four Abstract
ZorO (Z-protein often repeated) is a small protein expressed by pathogenic enterohemorrhagic Escherichia coli (EHEC E. coli) which was recently discovered through bioinformatic searches. While its biological role is still being elucidated, it has been demonstrated to exhibit toxic effects on the cell when overproduced. This puzzling genetic ability for what, at first glance, appears to be self-destruction may be nuanced way for wild strains of bacteria to adapt to host environments or survive stress. This likely entails a much more minimal expression of ZorO, initiating subtle cell behavior that is less apocalyptic. In order to investigate the localization and function of endogenously expressed ZorO, the protein was be synthesized for biophysical experimentation using solid-phase peptide synthesis. In addition, several analogs of ZorO were synthesized to further enable an investigation of structure-activity relationship. These analogs include versions of ZorO with single amino acid mutations, as well as variants with “tags” for detection, purification, or capturing transient interactions. In the latter case, an unnatural replacement of the amino acid methionine (photo-methionine) was synthesized for incorporation into a synthetic peptide analog. This synthesis work required exploration of literature and reaction optimization, ultimately leading to a novel synthesis of photomethionine. Much of the synthetic groundwork for studying ZorO has now been laid, and efforts are underway to develop analytical methods for detecting ZorO in cell extracts—a daunting task considering the complex protein composition of the cell and the wide concentration ranges spanned by endogenously-expressed polypeptides
