Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

Reptiles can often experience perturbations that greatly influence their metabolic status (e.g., temperature, exercise, digestion, and ontogeny). The most common cause of fluctuations in metabolic status in post-embryonic reptiles is arguably digestion and physical activity (which will be further referred to as exercise). The objective of this thesis is to determine the mechanisms involved in CO2 transport during digestion, determine the mechanisms that allow for the maintenance of acid-base homeostasis during digestion, and observing the effect of an understudied form of exercise in semi-aquatic reptiles on the regulation of metabolic acidosis and base deficit. This dissertation provided evidence for potentially novel and under investigated mechanisms for acid-base homeostasis (e.g., small intestine and tissue buffering capacity; Chapters 3 & 4), while also debunking a proposed hypothesis for the function of an anatomical feature that still remains a mystery to comparative physiologist (Chapter 2). This thesis is far from systematic and exhaustive in its approach, however, the work accomplished in this dissertation has become the foundation for multiple distinct paths for ecologically relevant investigations of the regulation of metabolic acidosis/alkalosis in reptiles.

Microalgae being photosynthetic and having quick growth cycles can prove to be excellent candidates as biofactories for the production of aromatic compounds like para-hydroxybenzoic acid (pHBA) that act as a monomer in liquid crystal polymers. We developed transgenic lines of the model alga Chlamydomonas reinhardtii by performing nuclear transformation using electroporation. The transgenic cell lines expressed the ubiC gene that utilized chorismate from the shikimate pathway as a substrate to produce pHBA. The maximum yield of pHBA measured in these lines was 80 mg/L. Accruing pHBA can be toxic to the cells and the mechanism by which C. reinhardtii could detoxify pHBA is not known. C. reinhardtii genome was thus scanned for sequences similar to UDP-glucosyltransferase (UGT) that can transfer the glucose moiety to pHBA, rendering it non-toxic to the cell lines. Our analysis suggested the absence of any potential UGTs that could glycosylate pHBA and detoxify it. We further performed feeding experiments to test the ability of wt-type C. reinhardtii cells to detoxify pHBA and understand its fate. C. reinhardtii cells were fed with varying concentrations of pHBA and harvested at different time intervals. The HPLC chromatograms indicated a majority of the pHBA was catabolized. Based on these results, …

Trait-based approaches are useful in ecological research because of their potential ability to predict species responses from patterns present in the community and to infer mechanisms driving community assembly. Current approaches for fishes are lacking traits across all five fundamental niche dimensions (i.e. habitat, life history, trophic, metabolic and defense). This study quantified a broad range of fish functional traits across all five niche dimensions (commonly used traits and novel traits), quantified intra- and interspecific variation for each trait, tested for relationships among traits within and among niche dimensions, tested for phylogenetic conservatism of traits and assessed trait-environment relationships for a subset of these traits under two different contexts. Approximately one third of the quantified traits exhibited greater intraspecific variation than interspecific variation and were not included in subsequent analyses. There were similarities between phylogeny and trait dendrograms for all traits, and habitat, metabolic and defense traits. The traits identified in chapter 2 were able to explain species responses during different flow periods in two intermittent streams as well as species-specific differences in host microbiome at the onset of drought in one intermittent stream. The novel traits identified in chapter 2 did contribute to our understanding of the community assembly …

In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD …

The purpose of this study was to investigate the effects of Pb and hypoxia co-exposure on Pb toxicity and Ca homeostasis in early life stage (ELS) zebrafish (Danio rerio). Previous evidence indicates that exposure of ELS zebrafish to hypoxia (~20% air saturation) reduces Ca uptake, likely through down-regulation of the apical epithelial Ca channel (ECaC). Considering that Pb and Ca are known antagonists and compete for uptake pathways, it was hypothesized that co-exposure of Pb with hypoxia would decrease Pb toxicity by reducing Pb uptake (likely mediated through a reduced number of ECaCs). However, it was shown that at 96 hpf, whole body accumulation of both Pb and Ca was lower at 40% air saturation compared to 100% and 20% air saturation. This result closely aligned with the 96h LC50 results which showed the highest mortality of zebrafish at 40% compared to the other air saturation levels. This suggests that toxicity is likely the result of exacerbated hypocalcemia at 40% air saturation due to both Pb competition for Ca binding to Ca uptake channels/transporters, such as ECaC, and potentially reduced expression of such channels/transporters in response to this level of hypoxia. Overall, it appears that ELS zebrafish respond differentially to …

We investigated the hypothesis that exposure to inhaled diesel exhaust PM can alter the gut microbiome and intestinal integrity, thereby promoting systemic inflammatory response and early CVD risk, which are exacerbated by HF diet. Furthermore, we investigated whether the observed exposure and diet-mediated outcomes could be mitigated through probiotic treatment. We performed an exposure study on C57Bl/6 male mice, placed on either a low fat (LF) diet or a high-fat (HF) diet, and exposed via oropharyngeal aspiration to 35 μg diesel exhaust particles (DEP) suspended in 35 μl of sterile saline or sterile saline controls (CON) twice a week for four weeks. A subset of mice on HF diet were dosed with 0.3 g/day (PRO, ~7.5x108 CFU/day) of probiotic Ecologic® Barrier 849 (Winclove Probiotics) in drinking water during the course of the study. For our first aim, we investigated the alterations in the gut microbiome, measured circulating cytokines and lipopolysaccharide (LPS), and measured CVD biomarkers in the heart. Our results revealed that exposure to inhaled DEP results in gut dysbiosis characterized by expansion of the phyla Verrucomicrobia and Proteobacteria and reduction in Actinobacteria, which was exacerbated by HF diet. Probiotics mitigated the DEP-mediated expansion of Proteobacteria and re-established Actinobacteria in …

Probiotics/prebiotic supplementation represents a viable option for addressing systemic inflammation and chronic disease risk resulting from excessive body weight. The purpose of this feasibility study was to determine if 90-d of supplementation with prebiotic and probiotic could alter mRNA responsible for inflammation and subsequently metabolic health in weight stable overweight adults. Participants were advised to not change their diet or exercise habits during the study. All protocols were approved by the University IRB and participants gave written informed consent. Participants were randomly assigned to either placebo (N=7; rice flour) or combined (N=8) prebiotic (PreticX® Xylooligosaccharide; 0.8 g/d; ADIP) and probiotic (MegaDuo® Bacillus subtilis HU58 and Bacillus coagulans SC-208; 3 Billion CFU/d) and measurements were made at baseline, 30, 60, and 90-d. Whole body DXA scans (GE iDXA®) and blood 574-plex mRNA analysis (Nanostring®) were used to generate primary outcomes. Compared to placebo, supplementation was associated with a 36% reduction in visceral adipose tissue (p = 0.001). Supplement resulted in significant, differential expression of 15 mRNA associated with adipose tissue inflammation, systemic inflammation, and/or chronic disease risk. The key findings support that 90-d prebiotic/probiotic supplementation may be associated with an improved metabolic health, reduced adipose tissue inflammation, reduced systemic inflammation, and …