• Atp-Dependent Chromatin Remodeling Complexes In Xenopus Development

      Brown, Elvin E.; Krebs, Jocelyn E.; Drew, Kelly (2010)
      A central question in the study of vertebrate development is how to account for the exquisite interplay of genes within differentiating cells and of groups of cells as they create the organs of the vertebrate embryo. Recently it has become clear that gene regulation by epigenetic processes adds a formerly unappreciated level of complexity to the regulatory network of development. One form of epigenetic gene regulation is embodied in ATP-dependent chromatin remodeling complexes, which use the energy of ATP hydrolysis to alter the interactions of DNA and histones. Chromatin remodeling complexes can both promote and repress expression of a gene at the appropriate time and place in vertebrate development. The list of their known roles in development is long and growing. Here I have studied the developmental role of CHRAC17, a subunit of the CHRAC and ATAC complexes, by visualizing its expression and by ablating CHRAC17 function in Xenopus laevis embryos. Whole mount in situ hybridization localized CHRAC17 expression to the neural tube, cranial placodes, and myotomes. Loss of CHRAC17 function following injection of embryos with CHRAC17-specific morpholino oligonucleotides resulted in abnormal development in the neural tube, eyes, notochord, and pharyngeal pouches, underlining the critical importance of CHRAC17 function in Xenopus development. Similarly, ablating the function of CHD4, the ATPase motor of the NuRD chromatin remodeling complex, resulted in severe developmental abnormalities in early Xenopus development.
    • Ceramide Metabolism Regulates A Neuronal Nadph Oxidase Influencing Neuron Survival During Inflammation

      Barth, Brian M. (2009)
      Inflammation is a major component of acute and chronic pathologies of the central nervous system, including psychiatric disorders. Microglia respond to pathogens, injury, and toxins by secreting inflammatory mediators including pro-inflammatory cytokines in an event known as neuroinflammation. This thesis research investigated a link between neuroinflammation and oxidative stress, and ultimately neurodegeneration. The cytokine tumor necrosis factor alpha was shown to stimulate a neuronal NADPH oxidase (NOX), specifically by stimulating the production of ceramide and ceramide-1-phosphate via Mg 2+-neutral sphingomyelinase (Mg2+-nSMase) and ceramide kinase. Intriguingly, glucosylceramide blocked NOX activation, linking ceramide neutralization directly to a decline in oxidative stress. Most importantly, NOX activity interfered with actin and sphingosine kinase-1 via oxidation, demonstrating a positive and detrimental feedback mechanism that impedes neuronal survival pathways. Interestingly, crude extracts from wild Alaskan bog blueberries showed the ability to interfere with Mg2+-nSMase, demonstrating a specific neuroprotective property of the berry. Altogether, this thesis research defined a key neuronal pathway linking inflammation to oxidative stress via ceramide metabolism, potentially allowing for future therapeutic development to improve neuronal function and survival.
    • Culturability, Temporal Change, Phylogenetic Analysis, And Yield Of Bacterial Communities In A Subarctic Lake: Harding Lake

      Zhao, Xiaoming; Button, Don (2005)
      Heterotrophic bacteria, adapted to small concentrations of substrate, are a main component of the microbial flywheel. However, understandings of their activity, isolation, genetics, and nutrition are restricted to the large, easily isolated and culturable bacteria. By using the dilution culture method, apparent culturabilities could approach 10% in unamended lake water and were inversely proportional to the number of cells inoculated from mixed species in a natural environment from Harding Lake. Substrate additions could not improve bacterial culturability in the dilution cultures. Comparative sequence analyses of 16S rDNA genes showed that all bacterial species have similar lengths in the phylogenetic tree, suggesting similar evolution rates. These indicated close relationships among the six bacterial divisions: alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, cytophaga/flexibacter/bacteriodes, acidobacteria, and cyanobacteria. Possible reasons include that metabolic enzymes of these bacteria were modified to adapt to low temperatures from tropical temperatures in arctic areas at the same time. These findings may provide insight into the recent evolution of the bacteria in near polar freshwater. Moreover, a high abundance of alpha-proteobacteria and gamma-proteobacteria was found in Harding Lake, suggesting high growth rates of these bacteria in the freshwater region. This is consistent with a rapid continuous shift in the distribution of dominant species observed in Harding Lake, according to the TRFLP, DGGE, and flow cytometry data. Our results also suggested that input of dissolved organic matter derived from terrestrial plants and soils, introduction of terrestrial bacteria, and bacteria themselves led to the bacterial species shifts associated with the seasonal change. Bacterial growth yield is used to measure this carbon conversion efficiency. However, bacterial growth yields have been seriously underestimated in previous studies. Our in situ values for bacterial growth yield from an amino acid mix were actually closer to 50% and 70% in active systems by using a modified, sensitive and accurate method and increased with the increase of temperature between 1�C and 6�C.
    • Molecular systematics and biogeography of long-tailed shrews (Insectivora: Sorex) and northern flying squirrels (Rodentia: Glaucomys)

      Demboski, John Richard; Cook, Joseph A. (1999)
      Insight into phylogenetic and biogeographic relationships among several mammalian taxa in western North America was provided with DNA sequences of two mitochondrial genes (cytochrome b and ND4). Members of two species complexes of long-tailed shrews (genus Sorex ) and northern flying squirrels (genus Glaucomys) were examined, and a common theme of responses to past climate change and glacial cycles was evident. Diversification events indicated by the DNA sequences provide new perspectives regarding the deep and shallow history of these taxa. Analysis of seven species of the Sorex cinereus complex (and related species) revealed two major clades within the complex, Northern and Southern. These generally corroborate proposed morphological relationships and correspond to broadly defined habitat affiliations (xeric and mesic), respectively. Within the Northern clade, amphiberingian species represented a monophyletic group suggesting Beringia was a center of endemism. Next, five species of the S. vagrans complex and related species were assessed. Significant molecular variation was revealed that does not correspond to morphological differences within the complex. Two major clades within S. monticolus were observed, a widespread Continental clade (Arizona to Alaska, including S. neomexicanus) and a restricted Coastal clade (Oregon to southeast Alaska, including S. bairdi and S. pacificus). A regional examination of genetic variation in the northern flying squirrel in southeast Alaska was also performed. Results suggested that southern islands in the Alexander Archipelago were the result of recent colonization (founder event). Finally, a comparative phylogeographic analysis of a reduced data set (S. monticolus), a molecular data set for the American Pine Marten, Martes americana, and other published molecular studies were used to reexamine the role of glacial refugia in the biogeography of the north Pacific coast. Previous ideas regarding purported refugia may be overstated and may be the result of limited geographic sampling. This thesis provides new perspectives on processes (e.g., post-glacial colonization) driving mammalian phylogenetic and biogeographic structuring in western North America.
    • The Effect Of Mitochondrial Ultrastructure On Function And Thermal Tolerance In Antartic Notothenioid Fishes

      Mueller, Irina Andrea; O'Brien, Kristin; Taylor, Barbara E.; Kuhn, Thomas B.; Staples, James F. (2012)
      The loss of hemoglobin in Antarctic icefishes is correlated with high mitochondrial volume densities and altered mitochondrial morphology in their oxidative muscle compared to red-blooded Antarctic notothenioid fish species. We hypothesized that differences in mitochondrial morphology between icefishes and red-blooded species might be correlated with differences in mitochondrial properties at their habitat temperature, near 0�C. We further hypothesized that differences in function might become more pronounced as temperature increases and might contribute to the lower thermal tolerance of icefishes compared to red-blooded species. Proton leak, rates of reactive oxygen species (ROS) production, membrane susceptibility to peroxidation and levels of antioxidants were measured in mitochondria isolated from hearts of the icefishes Chaenocephalus aceratus and Chionodraco rastrospinosus, and the red-blooded species Gobionotothen gibberifrons and Notothenia coriiceps. In addition, levels of oxidized proteins and lipids, and antioxidant levels were quantified in oxidative muscles of icefishes and red-blooded species exposed to their critical thermal maximum (CTmax) - an acute, short-term heat stress, and in animals exposed to 4�C for one week. Rates of ROS production increased as temperature increased in mitochondria isolated from both white- and red-blooded fishes. Yet, isolated mitochondria of icefishes are less protected against ROS. Antioxidant levels normalized to phospholipid content were lower in icefishes compared to red-blooded species, suggesting that icefishes might be more likely to experience oxidative stress as temperature increases. These findings were supported by measurements made in animals exposed to their CTmax. Levels of oxidized lipids increased in hearts of both icefishes, while levels of oxidized proteins increased only in C. aceratus in response to exposure to CTmax. In contrast, neither levels of oxidized lipids nor proteins increased in red-blooded fishes in response to exposure to CTmax. Similarly, levels of oxidized proteins did not increase in C. rastrospinosus or N. coriiceps in response to exposure to 4�C. Antioxidant levels tended to be lower in icefishes compared to red-blooded species and did not increase in any species in response to exposure to CTmax or 4�C. In summary, our findings suggest that icefishes are more vulnerable to heat-induced oxidative stress compared to red-blooded fishes.