Over the past half century, obesity has become a widespread concern to human health. Obesity is defined as having a body mass index above 30 and is accompanied by altered metabolic physiology. Obesity is one of the leading causes of mortality in the United States and in the world. The prevalence of obesity has increased over the past few decades. High body fat in adipose, excess calorie intake, low aerobic fitness are all associated with the comorbidities of obesity. Recently, an emphasis has been given to increased leukocytes in adipose tissue and the resulting low-grade systemic inflammation on the development of insulin resistance. Unfortunately, the immune-metabolic molecular mechanisms by which increased inflammation leads to diseases is not fully understood. We studied Nucb2, a highly expressed gene in immune cells and encodes for nesfatin-1, a peptide reported to be a satiety signal. We saw that Nucb2 expression is increased in leukocytes of high fat diet (HFD) fed animals. Contrary to our hypothesis, Nucb2-/- animals showed no effect on food intake. We then performed euglycemic hyperinsulinemic clamp studies in wild type and Nucb2 mice on a chow diet and a HFD to determine insulin sensitivity. Interestingly, knocking out the Nucb2 gene significantly impaired insulin sensitivity only under HFD conditions. These experiments demonstrate that Nucb2 is key player in glucose homeostasis in obesity. We discovered macrophages deficient of Nucb2 impact the proinflammatory macrophages. Macrophages lacking Nucb2 increased proinflammatory cytokine production. This suggests that Nucb2 intrinsically regulates the inflammatory cytokine production cascade. Mechanistically, Nucb2 mediates its effects by increasing proinflammatory cytokine expression via down-regulation of NFκB signaling in leukocytes. In the absence of Nucb2, NFκB activity is increased in macrophages. In contrast, inhibiting NFκB caused the opposite response. Finally, in response to endotoxemia, Nucb2 is required to control the production proinflammatory cytokines. Collectively, these data highlight a novel mechanism whereby Nucb2 serves as a key mediator of immunometabolic control of inflammation and insulin resistance. Overall, the studies I have performed identified a novel mediator of the immune-metabolic cross talks in the context of obesity-induced insulin resistance.

In a world of rapidly changing environmental conditions, species must effectively respond to their changing habitat or risk extinction. The goal of my dissertation is to elucidate the origins and mechanisms underlying the recent successful population expansion of the invasive coral Oculina patagonica in the Mediterranean Sea. To do this, I have utilized nuclear markers and next-generation sequencing data for the coral host and its algal symbiont as well as environmental data. Although only recently first described from the waters of the Mediterranean, genetic, historical demographic, and fossil evidence suggests that O. patagonica has not been anthropogenically introduced from the western North Atlantic. Instead, my results support the hypothesis that Oculina spp. has had a long history in the eastern Atlantic but remained undetected until it recently began expanding in the Mediterranean to invasive numbers, likely in response to environmental changes. Next, I found that the symbiotic algal communities harbored by Oculina corals vary geographically, and that this variation does not match the geographical variation of the hosts genetics. Instead, sea surface temperature is better correlated to symbiotic community, particularly in the Mediterranean, which may reflect acclimatization to local thermal conditions. Finally, in a closer inspection of a rapid poleward range expansion of O. patagonica along the Spanish Mediterranean coast, I found increased genetic diversity and adaptation to temperature that may have promoted its success. Together, my dissertation chapters shed light on the mechanisms that have allowed a coral to be successful despite stressful and changing environmental conditions. Unlike many previous studies aimed at assessing the adaptive capabilities and long-term success of tropical corals, my dissertation focusses on the success and adaptive potential of an understudied temperate coral. The findings presented here support the knowledge that O. patagonica is able to successfully respond to changing environmental conditions in its native range via both symbiont switching and host adaptation.

In view of its mountainous terrain, peninsular shape, and status as an agglomeration of formerly separate landmasses, the island of Sulawesi, Indonesia, presents a fertile opportunity for examining biogeographical processes. Previous work on Sulawesi primates and amphibians suggested the existence of distinct areas of endemism (AoEs), but the relevance of these zones to a broader set of taxa has not been widely investigated, nor the reasons for their existence fully explored. Here, we use population genetic analyses of the endemic Sulawesi shrew, Crocidura elongata, to assess biogeographical partitioning according to the putative AoEs. We uncover significant cryptic diversity within C. elongata that poorly aligns with the AoEs. Rather, we identify patterns consistent with the earliest divergence occurring along elevational gradients, and subsequent divergence evolving across the islands AoEs. Our results suggest that disparate forces have contributed to the diversification of Sulawesis vertebrate fauna and complement other studies in highlighting the extensive intra-island diversification that has occurred among small mammal faunas on the larger islands of Indo-Australia.

Retina damage or disease in humans often leads to reactive gliosis, preventing the formation of new cells and resulting in visual impairment or blindness. Currently, treatments are being developed to stimulate repair or replacement of lost retinal neurons by intravitreal injection of stem cells or retina precursors. Though improving, these therapies are inefficient and not yet capable of fully restoring vision. In contrast to mammals, the zebrafish retina is capable of spontaneous regeneration upon damage, using Müller glia (MG) derived progenitors. Understanding how zebrafish MG initiate regeneration may allow for the discovery of treatments that prompt mammalian retinas to regenerate. Here I show that inhibition of GABA signaling facilitates MG proliferation and initiation of retina regeneration. Using pharmacological and genetic approaches to disrupt neurotransmitter signaling, I demonstrate that GABAA receptor inhibition stimulates spontaneous regeneration in undamaged zebrafish retinas while GABAA receptor activation inhibits regeneration in damaged zebrafish retinas. This is the first evidence that neurotransmitters control retina regeneration in zebrafish through an evolutionarily conserved mechanism of neurogenesis.

The objective of this dissertation is to understand the functions of Escherichia coli YqjA and YghB, which are members of the conserved DedA/Tvp38 membrane protein family. YqjA and YghB are inner membrane (IM) proteins with multiple predicted membranespanning domain, sharing 62% amino acid identity and partly overlapping functions. Simultaneous in-frame deletions of these two genes in a strain named BC202 results in various phenotypes including cell division defects, temperature sensitivity, and sensitivity to drugs and alkaline pH. The cell division defect of BC202 is due to the inefficient secretion of periplasmic amidases by the twin arginine transport (Tat) pathway into the periplasm and drug sensitivity is due to the inefficient function of various drug efflux pumps. Both these phenotypes are related to the loss of proton motive force (PMF) in BC202. Overexpression of MdfA, a Na+-K+/H+ antiporter or growth in acidic media has the ability to rescue all the phenotypes of BC202. In addition, the ∆yqjA mutant (but not the ∆yghB mutant) is alkaline sensitive and overexpression of yqjA can restore growth at alkaline pH only when more than 100mM of sodium or potassium ions is present in the growth medium. Osmotic pressure augments the YqjA mediated growth at alkaline pH. Furthermore, charged amino acids are also essential for YqjA and YghB function that were previously shown important for various secondary transporters. Additionally, yqjA expression is higher at alkaline pH and increased expression of yqjA also required sodium/potassium salts above pH 9.0. The transcriptional regulator CpxR is required for the expression of yqjA at alkaline pH in the presence of Na+/K+. Based on these results, we suggest YqjA and YghB are osmosensing proton-dependent transporters required for E. coli drug resistance and alkaline pH tolerance.

Genomic imprinting is a mechanism that targets epigenetic modifications to regulate gene transcription to express a gene from only one of its two parental alleles. Imprinted genes are typically clustered together and are involved in developmental regulation of the fetus. The paternally expressed gene 3 (Peg3) domain represents one such imprinted gene cluster involved in fetal growth regulation and maternal caring behavior. The transcription and imprinting control of the Peg3 domain requires the transcription factor Yin-Yang 1 (YY1), a protein that plays important roles throughout development. The first part of this work explores evidence for the hypothesis that half a dosage of YY1 may be involved in controlling the transcription and imprinting of Peg3 in vivo. The results reveal that Yy1 most likely functions as a transcriptional repressor in this domain. The results also provide new evidence for bi-allelic expression of Peg3 in the mouse brain. Altogether, this indicates that the maternal allele of Peg3 is expressed and functional in specific areas of the brain, including the choroid plexus, paraventricular nucleus (PVN), and the supraoptic nucleus (SON). The observed bi-allelic expression pattern indicates either de-repression of the maternal allele of the known promoter or the presence of alternative promoters for the Peg3 locus. Therefore, the second part of this work demonstrates that several alternative promoters exist for Peg3. The results reveal that these alternative promoters display allele-, tissue-, and developmental stage-specific expression patterns. This suggests that the activity of these alternative promoters have been functionally selected features for the Peg3 imprinted domain during mammalian evolution. The third part of this work develops a novel methodology that detects alternative promoters for Peg3 by incorporating both 5 rapid amplification of cDNA ends (5RACE) and next-generation sequencing (NGS) techniques. The results indicate that this NGS-based 5RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters. Overall, the research presented in this dissertation advances our understanding of how the YY1 transcription factor is involved in controlling the Peg3 imprinted domain and how alternative promoters may contribute to the allele-, tissue- and developmental stage-specific, Peg3 expression patterns observed in the mouse.

Paternally expressed gene 3 (Peg3) is an imprinted gene that encodes a protein with twelve C2H2 zinc finger domains. Thus, PEG3 protein is predicted to serve as a transcription factor that may regulate other gene expression. Chromatin immunoprecipitation (ChIP) experiment revealed a list of genes that are bound by PEG3, supporting PEG3 is a DNA-binding protein. Genome wide expression analysis using wild type and Peg3 knockout mouse models further suggested that a large number of genes were up-regulated in Peg3 knockout model, including imprinted genes and some tissue-specific genes, suggesting that PEG3 may mainly function as a repressor for its downstream genes. It is well known that most imprinted genes are organized into clusters (imprinted domains) to share cis-acting elements which can regulate imprinted gene expression. However, in Peg3 imprinted domain, one of my studies suggested that two oppositely imprinted genes in Peg3 imprinted domain could interact through their gene products rather than shared cis regulatory elements. According to the results, paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor. Removing PEG3 resulted in elevated expression of Zim1, thus PEG3 should serve as a repressor for Zim1. ChIP experiment further suggested that PEG3 might repress Zim1 expression through SETDB1/KAP1-driven H3K9me3 mechanism. Subsequent ChIP-seq analysis using mouse embryonic fibroblast (MEF) cells further identified 16 target genes as PEG3 downstream genes. Interestingly, most of these genes showed high level of expression in oocyte, in which Peg3 is not expressed. Furthermore, qRT-PCR analysis confirmed that PEG3 mainly functions as a repressor for these downstream genes. In addition, electromobility shift assay (EMSA) and the luciferase reporter assay further demonstrated that PEG3 can directly bind to H19-ICR to repress H19 expression. Overall, the research presented in this dissertation advances our understanding of the repression function of PEG3 protein and its potential tumor suppressor function linked to its downstream genes.

Aerobic carbon monoxide (CO) oxidation is used by phylogenetically and physiologically diverse microorganisms inhabiting a variety of terrestrial and aquatic ecosystems. Activity assays, culture-based studies, and molecular-based approaches targeting the coxL gene, encoding the large subunit of CO dehydrogenase, were used to investigate the role of temperature in structuring CO-oxidizing communities at Kilauea Volcano, Hawaii. CO uptake activity was assessed for unvegetated and vegetated temperate volcanic deposits that experience different temperature regimes due to plant development during ecosystem succession. Both CO-oxidizing communities had similar short-term responses to temperature; however, results from extended incubations (30 d) at elevated temperature (55 °C) indicate that succession expanded the capacity of the vegetated community to adapt to high temperature. Aerobic CO uptake was also examined for geothermal sites including two soils and two microbial biofilms at the Puhimau geothermal area and Kilauea Iki crater. CO oxidation occurred at elevated temperatures for all sites assessed; however, cardinal temperatures for CO activity were not strongly correlated to in situ temperatures. These results also extended the known upper temperature limit (80 °C) for aerobic CO oxidation. Culture-based methods targeting thermophiles at these sites yielded 31 newly isolated thermophilic CO-oxidizing strains in 8 genera. Two strains were formally described as novel species. The isolation of multiple Thermogemmatispora strains (Class: Ktedonobacteria) led to exploration of CO oxidation in this group; CO oxidation was found to be a common trait among Ktedonobacteria. Additionally, geothermally-heated biofilms at Puhimau were dominated by Ktedonobacteria as determined from coxL clone libraries, 16S rRNA gene pyrosequencing, and analysis of coxL fragments from a biofilm metagenome. Thermophiles are known to exist in temperate environments, but their maintenance and activity remain unclear. We examined the activity of thermophilic CO-oxidizing bacteria under different temperature regimes including 25 °C, 55 °C, and an oscillating temperature regime (20 55 °C). Three hours per day above 45 °C was sufficient for growth and CO oxidation activity. CO oxidation at moderate temperatures could contribute to maintenance metabolism and survival of thermophiles under suboptimal conditions. Collectively these studies show that thermophilic CO oxidizers are active and abundant in thermal systems on Kilauea Volcano.

The greenhouse gas methane mainly regulated by microbial processes. Methanogenic archaea, a major biological source of methane, are a diverse group of organisms that produce methane as a metabolic byproduct. The generation of methane takes place in diverse anoxic environments using CO2 and other reduced substrates. The release of methane into oxic environments often is regulated by methanotrophic Proteobacteria and Archaea, specialized groups of bacteria that use methane as sole sources of carbon and energy. These organisms can play major roles in the regulation of methane transport into the atmosphere and make up one part of the methane cycle. Pools of are methane thought to be sequestered from interacting with the global methane cycle may exist beneath the worlds glaciers and ice sheets. These environments are cold, dark, and anoxic. Thereby these environments would be suitable for the growth and metabolism of methanogenic bacteria. Subglacial environments harbor active microbial ecosystems that potentially impact biogeochemical cycling and contribute significantly to global iron, sulfur, and carbon cycles. Recent molecular and biogeochemical evidence has suggested that subglacial environments are sources of the greenhouse gas methane. Here we present molecular and geochemical evidence of microbially derived subglacial methane being released at sites of subglacial discharge from a glacial terminus near Kangerlussuaq, Greenland. Dissolved methane in subglacial discharge was measured in samples collected during the summer of 2012 and ranged from 0.97 to 85 µM; δ13CH4 values for the methane indicated a biological origin. Duplicate samples that were not killed at time of collection showed depleted methane concentrations and heavy enrichment in δ13CH4 after four months of 4°C incubation. Molecular evidence, in the form of reverse transcribed pmoA mRNA and 16S rRNA, for active methanotrophs and recently active methanogens was found in these samples. Furthermore, a return visit to the subglacial site the following year provided additional evidence of an active methanotrophic community based on time series incubations (methane oxidation rates of 0.31 μM day-1). These data provide the first evidence suggesting that active microbial oxidation of subglacial methane is occurring at the western margin of the Greenland Ice Sheet.

This dissertation investigated the cellular and molecular basis of osmoregulation in developing and adult Gulf killifish, Fundulus grandis, acclimated to salinities ranging from fresh water to sea water. In chapter 2, F. grandis embryos were reared in 0.1, 5, and 32 ppt water from 2 days (d) post fertilization until late embryogenesis. There were no discernable differences among salinities in the morphology of osmoregulatory organs, including the pharyngeal arches, digestive tract, or kidney. The localization and abundance of Na+/K+ ATPase (NKA), Na+/K+/2Cl- cotransporter (NKCC), and the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) on the external surfaces of these tissues support a role in osmoregulatory as early as prior to hatch. In addition, F. grandis larvae were exposed from hatch to 0.1, 5, and 32 ppt water until 6 weeks post hatch (wph). Differential localization of NKCC and CFTR during osmotic challenges indicated putative altered functions of the intestine and gills, but not of the kidney in larval F. grandis. In Chapter 3, adult F. grandis were acutely transferred from 5 ppt water to 0.1, 1, and 5 ppt water for 7 d, or were acclimated to 0.1 ppt water then acutely transferred to 0.1 and 32 ppt water for 7 d. The mRNA and protein levels of NKA, NKCC, and CFTR in the intestine were differentially affected by the hypo- or hyperosmotic challenges. Localization of transport proteins suggested differences in intestine function associated with salinity acclimation. The anterior and posterior intestine may function in HCO3- or Cl- secretion during exposure to 0.1 ppt water; the anterior intestine from 32 ppt acclimated fish may facilitate both Cl- and water absorption; and the posterior intestine during exposure to 32 ppt water may facilitate HCO3- or Cl- secretion. In chapter 4, the roles of these proteins in the anterior intestine of F. grandis were assessed using an electrophysiological approach coupled with pharmacological inhibition of transporters. A high apical CFTR activity in the anterior intestine during acclimation in fresh water was observed. In conclusion, salinity acclimation affected the possible functions of osmoregulatory tissues of larval F. grandis, as well as the putative functions in ion/water regulation in the intestine of adult F. grandis.