The ability to acclimate to changing temperature has consequences for the biogeographic range of a species and their potential for surviving ocean warming. Using a tide simulator, which recreates tidal conditions by controlling water levels, water and air temperatures, light levels, and food availability, we explored how temperature and tidal zone (i.e. intertidal and subtidal) influences the abundance of sirtuin 5 (SIRT5) protein in a California native mussel (Mytilus californianus). We compared how gill tissue abundance of SIRT5, a key regulator of the cellular stress response and energy metabolism responded in M. californianus exposed to different temperature conditions (13, 16, 19 and 22°C) over a period of four weeks. Two SIRT5 isoforms, one a putative cytosolic form and the other a mitochondrial form were found to be expressed in mussel gill tissue. The mitochondrial isoform increased during acclimation to warm temperatures. This finding is the first to show how SIRT5 protein abundance changes with temperature acclimation. Surprisingly, we did not identify any differences in gill SIRT5 abundance between mussels from intertidal and subtidal locations. Our results suggest that characterizing the responses of SIRT isoforms may lead to a better understanding of the physiological diversity of sirtuins.

Aortic stiffness is an independent risk factor that has prognostic value regarding future cardiovascular disease (CVD) events such as myocardial infarction, strokes, and heart failure. Although death rates due to coronary heart disease have declined in recent years, the leading global killer remains CVD and prevalence is still high. Understanding lifestyle contributors associated with aortic stiffness would provide the public with insight into targeting key health-related behaviors. The purpose of this observational study was to examine the association of physical activity, physical function, and dietary quality as independent factors contributing to aortic stiffness in apparently healthy middle aged men. Fifty-two men between the ages of 30 and 59 years were recruited to participate in this study, which required two visits to the Exercise Physiology Laboratory. Aortic stiffness was measured by aortic pulse wave velocity (aPWV) and was not associated with total daily step counts (r=-0.06; P=0.70). However, aortic stiffness was inversely associated with physical function, determined with the sitting-rising test score (r=-0.44; P<0.01) and inversely associated with relative muscular strength, determined with peak handgrip strength in both hands normalized to body mass (r=-0.41; P<0.01). Additionally, aortic stiffness was inversely associated with dietary quality, determined with the Healthy Eating Index score (r=0.51; P<0.01). In conclusion, key health-related behaviors in this study that explained a large percentage of the variation in aortic stiffness were physical function and dietary quality (Adj r²=0.47; SEE=0.634). Hence, optimizing overall musculoskeletal fitness by focusing on strength, balance, coordination, and flexibility in addition to greater adherence to the U.S. Dietary Guidelines are key lifestyle contributors associated with reduced CVD risk in otherwise healthy middle aged men.

One hypothesis explaining the numerous independent evolutionary transitions from oviparity to viviparity among squamates (snakes and lizards) proposed that squamate embryonic development is independent of eggshell calcium. Recent research showed at least 25% of the calcium in hatchling oviparous squamates is extracted from the shell. Though not a direct test, these results are inconsistent with the hypothesis. To directly test the hypothesis, we removed eggshell calcium (through peeling) early in development of Pantherophis guttatus (corn snake) eggs. Survivorship to hatching did not differ between peeled and intact eggs. Yet hatchlings from peeled eggs were shorter (273.6 ± 3.4 vs. 261.0 ± 3.7 mm, p=0.0028, n=16), lighter (6.36 ±0.22 vs. 5.75 ± 0.23 g, p=0.0158, n=16), and had reduced calcium (40.8 ± 1.7 vs. 30.5 ± 1.8 mg, p

The aquatic zooplankton crustacean Daphnia magna must be able to tolerate thermal stress in order to survive their native shallow ponds that are susceptible to drastic seasonal and diurnal temperature fluctuations as well as to globally increasing temperatures. Survival in such variable environments requires plastic responses that must include fundamental aspects of Daphnia biochemistry and physiology. Adaptive response to selection favoring such plastic phenotypes requires the presence of genetic variation for plastic response in natural populations. Adverse effects of elevated temperature on aquatic organisms are diverse and so are their plastic responses; among the most severe challenges aquatic organisms face when exposed to heat is the elevated oxidative stress. In this work we focused on short-term and long-term responses of Daphnia to temperature changes that increase its resistance to oxidative stress. Daphnia acclimated to stressful but non-lethal temperature (28ºC) show longer survive during exposure to a lethal temperature (37ºC) than those acclimated to the optimal temperature (18ºC). Short-term reciprocal switches between 18ºC and 28ºC result in intermediate temperature tolerance. These changes are accompanied by mirroring changes in total antioxidant capacity indicating the increased antioxidant capacity as a possible causative mechanism for heat tolerance gained from acclimation. The analysis of 6 geographically distinct genotypes representing a range of temperature tolerance levels shows a genetic difference in response to short-term and long-term acclimation as well as in the effect of antioxidant capacity on temperature tolerance. These results indicate a significant degree of local adaptation in heat and oxidative stress defenses in Daphnia and provide a better understanding of adaptive responses of this zooplankton crustacean to rising temperatures.

Baroreflex sensitivity (BRS) is often reduced in elderly populations and patients with chronic cardiovascular diseases leading to a concomitant rise in blood pressure variability (BPV) that is associated with increased cardiovascular related morbidity and mortality. Thus, there is a need to better understand the mechanisms by which BPV causes cardiovascular end-organ damage. Animal studies using sinoaortic denervation (SAD) to increase BPV have demonstrated pathologic changes in the structure of the heart and blood vessels; however, there is a paucity of data investigating changes in functional measures of the heart and smaller, resistance type arteries. Furthermore, the pathogenic mechanisms involved in BPV-induced cardiovascular end-organ damage remain unknown. Baroreceptor denervation results in multiple cardiac stressors, many of which are associated with production of reactive oxygen species. Oxidative stress is known to promote cardiovascular end-organ damage but it is unclear if it plays a role in models of increased BPV. Thus, this study was designed to investigate the functional responses of smaller resistance type arteries and the heart to chronic exposure to enhanced BPV. In addition, the role of oxidative stress on these functional responses in a normotensive rat model of increased BPV was also investigated. Rats were subjected to either SAD surgery or a sham procedure and were observed for six weeks. To determine the role of oxidative stress, SAD rats were either treated with the superoxide dismutase mimetic tempol or left untreated. During the observation period, mean blood pressure remained normotensive, whereas baroreflex sensitivity was reduced and BPV increased two to three fold. Weekly in vivo assessment of vascular function of the long posterior ciliary artery (LPCA) demonstrated a significant reduction in endothelial-dependent dilation starting three weeks after SAD surgery compared to the sham group. Endothelial-independent dilation was not affected by SAD. Structural changes were not evident in the LPCA following SAD. However, structural (wall thickness, wall area, and wall area/lumen area ratio) and functional (strain and distensibility) changes were observed in the aorta. Cardiac structural (hypertrophy) and functional (diastolic dysfunction) effects were also evident following six weeks of increased BPV. Antioxidant treatment with tempol did not have any effect on the SAD-induced increase in BPV or decrease in BRS. Nevertheless, chronic tempol treatment prevented or reduced the cardiovascular end-organ damage (endothelial-dependent vascular dysfunction, decreased aortic distensibility, cardiac and vascular hypertrophy, and cardiac dysfunction) observed in the untreated SAD group. These findings suggest that the pathology observed following SAD is at least partly mediated by oxidative stress. Antioxidant treatment in patients with increased BPV (e.g., hypertension, diabetes, heart failure) may prevent or ameliorate cardiovascular end-organ damage and reduce the overall risk for cardiovascular disease events.

Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced by the fermentation of leucine by colonic bacteria. IVA activates G-protein coupled receptors such as FFAR2, FFAR3, and OR51E1 known to be expressed on enteric neurons and enteroendocrine cells. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic propulsion; however, the effect of branched chain fatty acids on gastrointestinal motility is unknown. We hypothesize that IVA relaxes smooth muscle in a cAMP/PKA dependent manner by direct action on smooth muscle cells. IVA will also decrease peristalsis and encourage retention of luminal contents. This thesis investigates the effect of IVA on smooth muscle tension and peristaltic activity in isolated colon and individual smooth muscle cells. Colon segments from C57BL/6J mice were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with 10 μM acetylcholine (ACh) and the effects of IVA at several concentrations were measured in the absence and presence of Nitric Oxide Synthase inhibitor L-N-nitroarginine (L-NNA), neuronal action potential inhibitor tetrodotoxin (TTX), and adenylate cyclase inhibitor SQ22536. To study individual live cells, mouse smooth muscle was isolated from colon, suspended in smooth muscle buffer, and after contraction with ACh were relaxed with micromolar concentrations of IVA. For peristalsis studies, whole colonic segments isolated from C57BL/6J were catheterized and placed horizontally in organ baths with circulating Krebs buffer. The colon was clamped on the anal end, and a solution (5 μL per mm of colon length) of either Krebs buffer or 50 mM IVA was delivered from the oral end to the lumen. Video of the peristalsis was then analyzed for diameter, changes in diameter, velocity of diameter changes along the length of the colon, normalized to the anatomical changes in the proximal region. IVA in concentrations of 10 mM to 50 mM relaxed the ACh-induced contraction in a sigmoidal fashion. In separate studies, L-NNA nor TTX affected the ability of IVA to inhibit relaxation. SQ22536 inhibited IVA induced relaxation in longitudinal colon compared to vehicle control. In isolated cells, SQ22536 and PKA inhibitor H-89 inhibited IVA-induced relaxation. In peristalsis studies, 50 mM IVA in Krebs buffer changed the character of the peristaltic action by increasing proximal diameter, inhibiting contractions in the proximal end of the colon, and decreasing overall velocity of peristaltic contractions in the proximal region. The data indicate that the branched chain fatty acid IVA causes a concentration-dependent relaxation of colonic smooth muscle that is direct to the smooth muscle and independent of neuronal activity. This relaxation is cAMP/PKA dependent. In addition to the direct relaxation of smooth muscle, intraluminal IVA decreased overall colonic propulsive activity and encouraged retention of the luminal contents. We conclude that the ingestion and production of branched chain fatty acids could affect overall GI motility and is an area for study in dietary and therapeutic control of bowel activity.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease leading to decreased lung volume and eventual respiratory failure. At present, the median post-diagnosis lifespan is between three and six years. Myofibroblasts are collagen-secreting cells essential for wound healing, but also implicated in the fibroproliferation and extra cellular matrix deposition commonly seen in IPF. The nitric oxide (NO) signaling pathway is implicated in protomyofibroblast to myofibroblast transition and regulation. Previous work has shown that in pulmonary myofibroblasts, endothelial nitric oxide synthase (eNOS) is the primary NOS isoform expressed. The current study used cultured rat pulmonary myofibroblasts between passages two and five as a cell model. The cells were grown in normal growth media (DMEM + 10% FBS) or serum starved (DMEM + 0% FBS) to induce cellular differentiation. In this study, immunocytochemistry was used to show localization of eNOS is dependent on cellular differentiation, with protomyofibroblasts expressing eNOS primarily in the nucleus and protomyofibroblasts expressing eNOS in the perinuclear region. We also show catalytic activity and localization of eNOS are correlated by visualizing nitric oxide production in the cells using a permeable fluorescein chromophore. By using western blot analysis on fractionated cell lysates we found eNOS expressed in the nucleus under normal growth conditions. eNOS is at least partially regulated by intracellular calcium (Ca2+) and calmodulin (CaM). Western blot analysis using native eNOS and phospho-specific eNOS antibodies on fractionated cells treated with the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate (PMA) with and without addition of its antagonist ethylene glycol tetraacetic acid (EGTA) was conducted to investigate PKC’s role in eNOS regulation by phosphorylation. Indeed, PKC activation was found to mitigate expression in the nucleus, while inhibition of the activator restored the activity expression above basal levels. This finding correlates with previous data from our lab showing a decrease in activity in myofibroblasts treated with PMA and assayed amperometrically with an NO electrode.

Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, serves as a model organism to study the circadian clock in plants and animals. Rhodopsins are blue/green-light photoreceptors also found in C. reinhardtii. Chlamyrhodopsin (COP), the most abundant eyespot protein, was reported to have no role in the phototactic and photophobic responses in C. reinhardtii. Its function is yet unknown. In the present study, we hypothesized that the function of COP is to mediate entrainment of the circadian clock by light. In order to test this hypothesis, a C. reinhardtii selection marker conferring resistance to the antibiotic paromomycin was cloned into a COP RNAi construct obtained from another lab. Firstly, the COP RNAi construct was restriction digested to linearize it. The linearized plasmid was then blunt ended with T4 DNA polymerase and dephosphorylated with phosphatase. The linearized fragment was ligated with the paromomycin resistance marker obtained by restriction digestion of the plasmid containing it and transformed into E.coli. The recombinant clones obtained were confirmed by restriction digests. Fusion regions and the orientation of the insert in the recombinant plasmid were confirmed by sequencing. An attempt was made to transform C. reinhardtii with the construct, but this was not successful. Future studies will be required to optimize the C. reinhardtii transformation method. Transformants with reduced COP amounts can then be tested for defects in resetting the clock after light pulses. This will determine whether chlamyrhodopsin is involved in the circadian input pathway or not. The results of the complete project are expected to contribute to our understanding of the circadian clock of many other organisms including humans.

Vagal tone, a measure of parasympathetic activity via the vagus nerve, is known to be associated with positive emotion because it promotes social engagement and self-soothing behavior. Heart rate variability (HRV), especially high frequency oscillation, is a direct measure of vagal tone, and has been used in previous studies to test the correlation between vagal tone and positive emotion. This study aims to determine if the two major oscillations of heart rate variability, high frequency (HF-HRV) and low frequency (LF-HRV) can predict pro-social behavior, which is classified as giving donations to charities. Baseline LF- and HF-HRV levels were recorded, and compared to how much each participant donated after watching several donation appeals by charities. Results showed there was no significant difference in either LF- or HF-HRV levels between donors and non-donors. A negative correlation was found between both LF and HF-HRV levels and donation behavior, contradicting research that predicts a positive correlation between the two variables. There was no significant effect of gender on donation amount. This research gives insight in to how physiological changes can affect psychological processes, such as emotional expression.

The microbes that inhabit the human body, our microbiota, greatly influence our physiology and propensity for disease. For instance, the gut microbiota metabolizes compounds from our diet to provide important nutrients. Similarly, the microbiota has the potential to impact drug response; directly by metabolizing drugs, or indirectly by providing metabolites to the host. The complexity of the mammalian microbiota, and the limited throughput of such models, prohibit a systematic interrogation of specific interactions between microbes and host drug response. Here, I use C. elegans and its bacterial diet as a suitable model with the scalability and genetic tractability to address these questions. In Chapter II, I describe host-bacteria-drug interactions involving the anti-pyrimidine drugs 5-FU and FUDR. In brief, we identified two main mechanisms by which bacteria affect the C. elegans response to anti-pyrimidines: (1) metabolic conversion into FUMP by uridine phospho-ribosyltransferase (upp) and (2) dietary supplementation of uracil. Chapter III will focus on a selective estrogen-receptor modulator, TAM, with no clear target in bacteria or C. elegans. I will describe my work characterizing a bacteria-dependent response to TAM involving fatty acid metabolism. Lastly, the Appendix will summarize my efforts to expand the sample space of tested host-microbe-drug interactions.