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• 1. [Article] Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus

  Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene ...

  Citation × Citation Citation Abstract Copy Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Close

• 2. [Article] The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes

  Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding ...

  Citation × Citation Citation Abstract Copy Title: The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes Author: Davidge, Brittney Marie Year: 2017 Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway. Title: The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes Author: Davidge, Brittney Marie Year: 2017 Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway. Close

• 3. [Article] Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i>

  The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of ...

  Citation × Citation Citation Abstract Copy Title: Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Romney, Amie Lynn Thomas Year: 2017 The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can "escape" entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus. In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus. Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause. Title: Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Romney, Amie Lynn Thomas Year: 2017 The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can "escape" entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus. In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus. Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause. Close

• 4. [Article] ImpL2 Represses Insulin Signaling in Response to Hypoxia

  xiii, 56 p. : ill. (some col.)

  Citation × Citation Citation Abstract Copy Title: ImpL2 Represses Insulin Signaling in Response to Hypoxia Author: Allee, John Paul, 1970- Year: 2011 xiii, 56 p. : ill. (some col.) Title: ImpL2 Represses Insulin Signaling in Response to Hypoxia Author: Allee, John Paul, 1970- Year: 2011 xiii, 56 p. : ill. (some col.) Close

• 5. [Article] The effect of plant growth regulators on the growth of Closterium moniliferum

  Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant ...

  Citation × Citation Citation Abstract Copy Title: The effect of plant growth regulators on the growth of Closterium moniliferum Author: Christensen, Cynthia Lehua Warnock Year: 1990 Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant growth factors. The growth promoters NAA and GA both increased growth when added to the media. The cell division regulator BAP (a synthetic cytokinin). also had a promotive effect on growth. Abscisic acid was found to be inhibitive to growth. Title: The effect of plant growth regulators on the growth of Closterium moniliferum Author: Christensen, Cynthia Lehua Warnock Year: 1990 Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant growth factors. The growth promoters NAA and GA both increased growth when added to the media. The cell division regulator BAP (a synthetic cytokinin). also had a promotive effect on growth. Abscisic acid was found to be inhibitive to growth. Close

• 6. [Article] Robustness of a model microbial community emerges from population structure among single cells of a clonal population

  Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution ...

  Citation × Citation Citation Abstract Copy Title: Robustness of a model microbial community emerges from population structure among single cells of a clonal population Author: Thompson, Anne W., Turkarslan, Serdar, Arens, Christina E., de Lomana, Adrian Lopez Garcia, Raman, Arjun V., Stahl, David A., Baliga, Nitin S. Year: 2017 Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population. Title: Robustness of a model microbial community emerges from population structure among single cells of a clonal population Author: Thompson, Anne W., Turkarslan, Serdar, Arens, Christina E., de Lomana, Adrian Lopez Garcia, Raman, Arjun V., Stahl, David A., Baliga, Nitin S. Year: 2017 Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population. Close

• 7. [Article] Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans

  Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood ...

  Citation × Citation Citation Abstract Copy Title: Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans Author: Zygmunt, Andrew Christopher Year: 1984 Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood flow rate Is dependent upon cell viscosity, which in turn is a consequence of cell volume and membrane deform-ability. Cell volume changes which Increase membrane deform-ability will thus potentially extend the limits of dehydration tolerance. It was found in R. catesbeiana and B. marinus that red blood cell Is maintain constant volume during dehydration. Cells in vitro Initially lose water, but then sodium, potassium and water move Into the cell. Cell viscosity within the physiologic range of hematocrits was higher In salt loaded non-regulating cell Is of B. marinus than In regulating isotonic cells. Title: Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans Author: Zygmunt, Andrew Christopher Year: 1984 Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood flow rate Is dependent upon cell viscosity, which in turn is a consequence of cell volume and membrane deform-ability. Cell volume changes which Increase membrane deform-ability will thus potentially extend the limits of dehydration tolerance. It was found in R. catesbeiana and B. marinus that red blood cell Is maintain constant volume during dehydration. Cells in vitro Initially lose water, but then sodium, potassium and water move Into the cell. Cell viscosity within the physiologic range of hematocrits was higher In salt loaded non-regulating cell Is of B. marinus than In regulating isotonic cells. Close

• 8. [Article] Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i>

  Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although ...

  Citation × Citation Citation Abstract Copy Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Close

• 9. [Article] Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans

  A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected ...

  Citation × Citation Citation Abstract Copy Title: Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans Author: Johnston, Michael H. Year: 1977 A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected temperatures were continuously recorded for 24 hours. A second group of turtles was acclimatized to 7.5-11.0˚C and natural photoperiods for 28 days, and subsequently tested in the same manner as the first group. Lastly, the turtles in the first group, after being acclimated to 3.0˚C and the previous artificial photoperiod for 14 days, were retested in the gradient. Title: Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans Author: Johnston, Michael H. Year: 1977 A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected temperatures were continuously recorded for 24 hours. A second group of turtles was acclimatized to 7.5-11.0˚C and natural photoperiods for 28 days, and subsequently tested in the same manner as the first group. Lastly, the turtles in the first group, after being acclimated to 3.0˚C and the previous artificial photoperiod for 14 days, were retested in the gradient. Close

• 10. [Article] Neurobiology of Seasonal Life-history Transitions

  Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such ...

  Citation × Citation Citation Abstract Copy Title: Neurobiology of Seasonal Life-history Transitions Author: Lucas, Ashley Rae Year: 2015 Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they regulate reproductive and feeding behavior, respectively. In this study, I asked if seasonal changes in AVT and/or NPY are concomitant with spring migration away from the breeding grounds, as male and female red-sided garter snakes (Thamnophis sirtalis parietalis) are transitioning from reproductive to non-reproductive behavior during this time. To address this question, I collected male and female snakes in different migratory stages during the spring and fall. Brains were processed for AVT and NPY immunohistochemistry and the total number of immunoreactive (-ir) cells quantified for each individual. As predicted, males had significantly more AVT-ir cells in the preoptic area and bed nucleus of the stria terminalis, brain regions important for courtship behavior, during the spring mating season compared to the fall. Females had significantly more AVT-ir cells in the preoptic area during the spring compared to the fall and, surprisingly, did not exhibit seasonal changes in NPY. In contrast, males had significantly more NPY-ir cells in the cortex, a region important for spatial memory, and in the posterior hypothalamus during the fall compared to the spring, which likely reflects increased feeding behavior during the summer foraging period. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, indicating that seasonal changes in these neuropeptides are not directly related to migration. I then asked if the observed seasonal changes in AVT and NPY in males and females are related to the transition from reproductive to non-reproductive states. Compared to courting males, non-courting males had significantly more AVT-ir cells in the supraoptic nucleus and more NPY-ir cells in the cortex. AVT- and NPY-ir cells did not differ between unmated and mated females. Collectively, my results suggest that AVT and NPY play a role in regulating seasonal transitions in male reproductive behavior, rather than regulating migration per se. Further, these data indicate that both AVT and NPY are regulating reproductive behavior differently in males versus females. These data provide the framework for future studies examining the mechanisms regulating transitions between reproductive, migratory and foraging behaviors. Title: Neurobiology of Seasonal Life-history Transitions Author: Lucas, Ashley Rae Year: 2015 Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they regulate reproductive and feeding behavior, respectively. In this study, I asked if seasonal changes in AVT and/or NPY are concomitant with spring migration away from the breeding grounds, as male and female red-sided garter snakes (Thamnophis sirtalis parietalis) are transitioning from reproductive to non-reproductive behavior during this time. To address this question, I collected male and female snakes in different migratory stages during the spring and fall. Brains were processed for AVT and NPY immunohistochemistry and the total number of immunoreactive (-ir) cells quantified for each individual. As predicted, males had significantly more AVT-ir cells in the preoptic area and bed nucleus of the stria terminalis, brain regions important for courtship behavior, during the spring mating season compared to the fall. Females had significantly more AVT-ir cells in the preoptic area during the spring compared to the fall and, surprisingly, did not exhibit seasonal changes in NPY. In contrast, males had significantly more NPY-ir cells in the cortex, a region important for spatial memory, and in the posterior hypothalamus during the fall compared to the spring, which likely reflects increased feeding behavior during the summer foraging period. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, indicating that seasonal changes in these neuropeptides are not directly related to migration. I then asked if the observed seasonal changes in AVT and NPY in males and females are related to the transition from reproductive to non-reproductive states. Compared to courting males, non-courting males had significantly more AVT-ir cells in the supraoptic nucleus and more NPY-ir cells in the cortex. AVT- and NPY-ir cells did not differ between unmated and mated females. Collectively, my results suggest that AVT and NPY play a role in regulating seasonal transitions in male reproductive behavior, rather than regulating migration per se. Further, these data indicate that both AVT and NPY are regulating reproductive behavior differently in males versus females. These data provide the framework for future studies examining the mechanisms regulating transitions between reproductive, migratory and foraging behaviors. Close

• 11. [Article] Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation

  Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations ...

  Citation × Citation Citation Abstract Copy Title: Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation Author: Cole, Jessica, Ferguson, Amanda, Segarra, Verónica A., Walsh, Susan Year: 2017 Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions. Title: Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation Author: Cole, Jessica, Ferguson, Amanda, Segarra, Verónica A., Walsh, Susan Year: 2017 Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions. Close

• 12. [Article] Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish

  Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, ...

  Citation × Citation Citation Abstract Copy Title: Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish Author: Hand, Steven C., Denlinger, David L., Podrabsky, Jason E., Roy, Richard Year: 2016 Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages. Title: Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish Author: Hand, Steven C., Denlinger, David L., Podrabsky, Jason E., Roy, Richard Year: 2016 Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages. Close

• 13. [Article] Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i>

  Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus ...

  Citation × Citation Citation Abstract Copy Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Close

• 14. [Article] Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus

  Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the ...

  Citation × Citation Citation Abstract Copy Title: Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus Author: Woll, S. Cody, Podrabsky, Jason E. Year: 2017 Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short postembryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus. Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes. Title: Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus Author: Woll, S. Cody, Podrabsky, Jason E. Year: 2017 Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short postembryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus. Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes. Close

• 15. [Article] Epigenetic Regulation of Alternative Developmental Trajectories

  The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an ...

  Citation × Citation Citation Abstract Copy Title: Epigenetic Regulation of Alternative Developmental Trajectories Author: Romney, Arnie Lynn T., Podrabsky, Jason E. Year: 2017 The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead "escaped" entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA-seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA-seq data during early development, after the maternal-to-zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal-embryonic-conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development. Title: Epigenetic Regulation of Alternative Developmental Trajectories Author: Romney, Arnie Lynn T., Podrabsky, Jason E. Year: 2017 The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead "escaped" entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA-seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA-seq data during early development, after the maternal-to-zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal-embryonic-conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development. Close

• 16. [Article] The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality

  The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant ...

  Citation × Citation Citation Abstract Copy Title: The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality Author: Campbell, James Dudley Year: 1981 The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant apparatus was designed to test metabolic output at different heat reinforcement magnitudes. The mean TB reflected in each trial was positively correlated to the length of reinforcement. The shuttle rate during each trial was inversely correlated to the length of reinforcement. The standard deviation and total metabolic costs did not vary significantly between trials undertaken at the same ambient temperature. Eurythermality in G. coeruleus is caused by fluctuations in preferred body temperature and not by fluctuations around this temperature. The metabolic cost of behavioral thermoregulation did not change with corresponding changes in reinforcement magnitude. This indicates that eurythermality is the recorded effect of lizards behaviorally regulating to different TB in a stenothermic manner, rather than of random TB fluctuations in a wide range of normal activity (the classical view of eurythermality). Title: The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality Author: Campbell, James Dudley Year: 1981 The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant apparatus was designed to test metabolic output at different heat reinforcement magnitudes. The mean TB reflected in each trial was positively correlated to the length of reinforcement. The shuttle rate during each trial was inversely correlated to the length of reinforcement. The standard deviation and total metabolic costs did not vary significantly between trials undertaken at the same ambient temperature. Eurythermality in G. coeruleus is caused by fluctuations in preferred body temperature and not by fluctuations around this temperature. The metabolic cost of behavioral thermoregulation did not change with corresponding changes in reinforcement magnitude. This indicates that eurythermality is the recorded effect of lizards behaviorally regulating to different TB in a stenothermic manner, rather than of random TB fluctuations in a wide range of normal activity (the classical view of eurythermality). Close

• 17. [Article] Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication

  A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers ...

  Citation × Citation Citation Abstract Copy Title: Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication Author: Wilczynski, Walter, Lutterschmidt, Deborah I. Year: 2016 A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers and provide a striking example of melatonin's niche-specific functions. Title: Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication Author: Wilczynski, Walter, Lutterschmidt, Deborah I. Year: 2016 A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers and provide a striking example of melatonin's niche-specific functions. Close

• 18. [Article] Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance

  Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet ...

  Citation × Citation Citation Abstract Copy Title: Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance Author: Riggs, Claire Louise Year: 2017 Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells. Title: Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance Author: Riggs, Claire Louise Year: 2017 Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells. Close

• 19. [Article] Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus

  The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic ...

  Citation × Citation Citation Abstract Copy Title: Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus Author: Chan, Eva Y Year: 2016 The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic axis. In addition, embryonic diapause can occur. Diapause II and III are often observed when embryos are incubated at 25oC. Maternal influences and incubation environment are the factors that determine entrance into embryonic diapause, but the molecular mechanisms that regulate diapause are still unknown. Interestingly, embryonic diapause and the unusual cell movements observed during early development are always found together in species of annual killifish that exhibit diapause. Bone morphogenetic proteins (BMPS) are crucial in the development of various embryonic tissues. A recent study found the gene expressions pattern of BMP antagonists, noggin, chordin and follistatin, in early - embryonic development in A. limnaeus are different from zebrafish. This finding suggests BMPs may play roles in the regulation of diapause. In the present paper, the expression of BMP-2, BMP-4 and the BMP Receptor-2 were examined during early embryonic development in A. limnaeus using reverse transcription polymerase chain reaction (RT-PCR). Title: Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus Author: Chan, Eva Y Year: 2016 The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic axis. In addition, embryonic diapause can occur. Diapause II and III are often observed when embryos are incubated at 25oC. Maternal influences and incubation environment are the factors that determine entrance into embryonic diapause, but the molecular mechanisms that regulate diapause are still unknown. Interestingly, embryonic diapause and the unusual cell movements observed during early development are always found together in species of annual killifish that exhibit diapause. Bone morphogenetic proteins (BMPS) are crucial in the development of various embryonic tissues. A recent study found the gene expressions pattern of BMP antagonists, noggin, chordin and follistatin, in early - embryonic development in A. limnaeus are different from zebrafish. This finding suggests BMPs may play roles in the regulation of diapause. In the present paper, the expression of BMP-2, BMP-4 and the BMP Receptor-2 were examined during early embryonic development in A. limnaeus using reverse transcription polymerase chain reaction (RT-PCR). Close

• 20. [Article] Effect of ethanol on thermoregulation in the goldfish, Carassius auratus

  In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was ...

  Citation × Citation Citation Abstract Copy Title: Effect of ethanol on thermoregulation in the goldfish, Carassius auratus Author: O'Connor, Candace Sharon Year: 1986 In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was administered to 10 to 15 g fish by mixing it in the water of a temperature gradient. The dose response curve was very steep between 0.5% (v/v) ethanol (no response) and 0.7% (significant lowering of selected temperature in treated fish). Fish were exposed to concentrations of ethanol as high as 1.7%, at which concentration most experimental fish lost their ability to swim upright in the water. At concentrations higher than 0.7%, the magnitude of the effect did not increase with increasing concentration of ethanol; treated animals continued to select temperatures about 2 C below temperatures selected by controls. Experiments alternating exposure to 1.0% ethanol and water showed that the rate of onset and disappearance of the ethanol effect was rapid (within 10 min). Other experiments exposing fish to 1.0% ethanol for up to 3 hr showed that the effect remained stable for this period of time. The thermoregulatory responses of fish are behavioral, and therefore relatively easy to observe and quantify. Ethanol produces a prompt, stable and reproducible depression of selected temperature in the goldfish. Because the temperature at which fish regulate is controlled by a central nervous system set point and not altered by effects on peripheral effector systems, it appears that ethanol may cause hypothermia in goldfish by directly acting to lower the set point. Title: Effect of ethanol on thermoregulation in the goldfish, Carassius auratus Author: O'Connor, Candace Sharon Year: 1986 In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was administered to 10 to 15 g fish by mixing it in the water of a temperature gradient. The dose response curve was very steep between 0.5% (v/v) ethanol (no response) and 0.7% (significant lowering of selected temperature in treated fish). Fish were exposed to concentrations of ethanol as high as 1.7%, at which concentration most experimental fish lost their ability to swim upright in the water. At concentrations higher than 0.7%, the magnitude of the effect did not increase with increasing concentration of ethanol; treated animals continued to select temperatures about 2 C below temperatures selected by controls. Experiments alternating exposure to 1.0% ethanol and water showed that the rate of onset and disappearance of the ethanol effect was rapid (within 10 min). Other experiments exposing fish to 1.0% ethanol for up to 3 hr showed that the effect remained stable for this period of time. The thermoregulatory responses of fish are behavioral, and therefore relatively easy to observe and quantify. Ethanol produces a prompt, stable and reproducible depression of selected temperature in the goldfish. Because the temperature at which fish regulate is controlled by a central nervous system set point and not altered by effects on peripheral effector systems, it appears that ethanol may cause hypothermia in goldfish by directly acting to lower the set point. Close

• 21. [Article] Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance

  Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out ...

  Citation × Citation Citation Abstract Copy Title: Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out of anoxia in species that can tolerate anoxia may lend new insights into the role that mitochondria play in cellular homeostasis in response to fluctuating levels of oxygen. The objective of this study was to examine the potential role of small RNAs in long-term vertebrate anoxia tolerance. Expression patterns of small RNAs were determined using RNAseq in the most anoxia tolerant vertebrate, embryos of the annual killifishAustrofundulus limnaeus. Anoxia tolerance varies during development in this species in a way that allows for the comparison of developmental stages with extreme anoxia tolerance to stages with a more typical vertebrate anoxia tolerance. Embryos of 4 different developmental stages were exposed to anoxia, followed by aerobic recovery, and small RNA expression was determined with Illumina RNAseq under aerobic conditions, anoxia, and during aerobic recovery. A large proportion of the differentially expressed small RNAs were found to be encoded in the mitochondrial genome. About 12% of the unique small RNAs identified were encoded within the mitochondrial genome, and around 20% of the highly differentially expressed RNAs in response to anoxia were mitochondrial in origin. Of the differentially expressed sequences mapping to the mitochondrial genome, small RNAs derived from mitochondrial tRNAs were particularly enriched, with sequences up-regulated during anoxia and aerobic recovery from anoxia. These small RNAs of interest localize to anoxia sensitive tissues, such as the brain and heart, providing further evidence for a potential role in supporting anoxia tolerance. These same small RNAs have been detected in cell cultures derived from A. limnaeus embryos, by RNAseq and in situ hybridization. Cell culture expression patterns corroborated whole-embryo sequence expression patterns. This work highlights a potential new class of small RNAs derived from the mitochondrial genome that may act to mediate and coordinate the physiological response to anoxia in this species. Further understanding the biology of these mitochondrially-derived small RNAs and their role in anoxia tolerance may provide novel insight into combating cellular damage induced by heart attack and stroke, two lethal consequences of heart disease in humans. Title: Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out of anoxia in species that can tolerate anoxia may lend new insights into the role that mitochondria play in cellular homeostasis in response to fluctuating levels of oxygen. The objective of this study was to examine the potential role of small RNAs in long-term vertebrate anoxia tolerance. Expression patterns of small RNAs were determined using RNAseq in the most anoxia tolerant vertebrate, embryos of the annual killifishAustrofundulus limnaeus. Anoxia tolerance varies during development in this species in a way that allows for the comparison of developmental stages with extreme anoxia tolerance to stages with a more typical vertebrate anoxia tolerance. Embryos of 4 different developmental stages were exposed to anoxia, followed by aerobic recovery, and small RNA expression was determined with Illumina RNAseq under aerobic conditions, anoxia, and during aerobic recovery. A large proportion of the differentially expressed small RNAs were found to be encoded in the mitochondrial genome. About 12% of the unique small RNAs identified were encoded within the mitochondrial genome, and around 20% of the highly differentially expressed RNAs in response to anoxia were mitochondrial in origin. Of the differentially expressed sequences mapping to the mitochondrial genome, small RNAs derived from mitochondrial tRNAs were particularly enriched, with sequences up-regulated during anoxia and aerobic recovery from anoxia. These small RNAs of interest localize to anoxia sensitive tissues, such as the brain and heart, providing further evidence for a potential role in supporting anoxia tolerance. These same small RNAs have been detected in cell cultures derived from A. limnaeus embryos, by RNAseq and in situ hybridization. Cell culture expression patterns corroborated whole-embryo sequence expression patterns. This work highlights a potential new class of small RNAs derived from the mitochondrial genome that may act to mediate and coordinate the physiological response to anoxia in this species. Further understanding the biology of these mitochondrially-derived small RNAs and their role in anoxia tolerance may provide novel insight into combating cellular damage induced by heart attack and stroke, two lethal consequences of heart disease in humans. Close

• 22. [Article] A Metabolic Hypothesis for the Evolution of Temperature Effects on the Arterial PCO2 and pH of Vertebrate Ectotherms

  Body temperature increases in ectothermic vertebrates characteristically lead to both increases in arterial PCO2 (PaCO2) and declines in resting arterial pH (pHa) of about 0.017 pH units/°C increase in ...

  Citation × Citation Citation Abstract Copy Title: A Metabolic Hypothesis for the Evolution of Temperature Effects on the Arterial PCO2 and pH of Vertebrate Ectotherms Author: Hillman, Stanley S., Hedrick, Michael Scott Year: 2017 Body temperature increases in ectothermic vertebrates characteristically lead to both increases in arterial PCO2 (PaCO2) and declines in resting arterial pH (pHa) of about 0.017 pH units/°C increase in temperature. This ‘alphastat’ pH pattern has previously been interpreted as being evolutionarily-driven by the maintenance of a constant protonation state on the imidazole moiety of histidine protein residues, hence stabilizing protein structure-function. Analysis of the existing data for interclass responses of ectothermic vertebrates show different degrees of PaCO2 increases and pH declines with temperature between the classes with reptiles>amphibians>fish. The PaCO2 at the temperature where maximal aerobic metabolism (VO2max) is achieved is significantly and positively correlated with temperature for all vertebrate classes. For ectotherms, the PaCO2 where VO2max is greatest is also correlated with VO2max indicating there is an increased driving force for CO2 efflux that is lowest in fish, intermediate in amphibians and highest in reptiles. The pattern of increased PaCO2 and the resultant reduction of pHa to increased body temperature would serve to increase CO2 efflux, O2 delivery, blood buffering capacity and maintain ventilatory scope. This represents a new hypothesis for the selective advantage of arterial pH regulation from a systems physiology perspective in addition to the advantages of maintenance of protein structure-function. Title: A Metabolic Hypothesis for the Evolution of Temperature Effects on the Arterial PCO2 and pH of Vertebrate Ectotherms Author: Hillman, Stanley S., Hedrick, Michael Scott Year: 2017 Body temperature increases in ectothermic vertebrates characteristically lead to both increases in arterial PCO2 (PaCO2) and declines in resting arterial pH (pHa) of about 0.017 pH units/°C increase in temperature. This ‘alphastat’ pH pattern has previously been interpreted as being evolutionarily-driven by the maintenance of a constant protonation state on the imidazole moiety of histidine protein residues, hence stabilizing protein structure-function. Analysis of the existing data for interclass responses of ectothermic vertebrates show different degrees of PaCO2 increases and pH declines with temperature between the classes with reptiles>amphibians>fish. The PaCO2 at the temperature where maximal aerobic metabolism (VO2max) is achieved is significantly and positively correlated with temperature for all vertebrate classes. For ectotherms, the PaCO2 where VO2max is greatest is also correlated with VO2max indicating there is an increased driving force for CO2 efflux that is lowest in fish, intermediate in amphibians and highest in reptiles. The pattern of increased PaCO2 and the resultant reduction of pHa to increased body temperature would serve to increase CO2 efflux, O2 delivery, blood buffering capacity and maintain ventilatory scope. This represents a new hypothesis for the selective advantage of arterial pH regulation from a systems physiology perspective in addition to the advantages of maintenance of protein structure-function. Close

• 23. [Article] Factors affecting thyrotropin secretion in superfused rat anterior pituitary cells

  The use of static in vitro pituitary cultures has been indispensible for examining the regulation of anterior pituitary hormone secretion. While the static cultures have shown the effects of various chemical ...

  Citation × Citation Citation Abstract Copy Title: Factors affecting thyrotropin secretion in superfused rat anterior pituitary cells Author: Bartlow, Frederick Scott Year: 1982 The use of static in vitro pituitary cultures has been indispensible for examining the regulation of anterior pituitary hormone secretion. While the static cultures have shown the effects of various chemical stimulators of hormone secretion, the dynamics of such secretion has not been assessed before in vitro. The in vitro superfusion method, which partially stimulates in vivo physiologic conditions, allows for the observation of the dynamics of pituitary hormone secretion. Title: Factors affecting thyrotropin secretion in superfused rat anterior pituitary cells Author: Bartlow, Frederick Scott Year: 1982 The use of static in vitro pituitary cultures has been indispensible for examining the regulation of anterior pituitary hormone secretion. While the static cultures have shown the effects of various chemical stimulators of hormone secretion, the dynamics of such secretion has not been assessed before in vitro. The in vitro superfusion method, which partially stimulates in vivo physiologic conditions, allows for the observation of the dynamics of pituitary hormone secretion. Close

• 24. [Article] The role of iron nutrition in regulating patterns of photosynthesis and nitrogen metabolism in the green alga Scenedesmus quadricauda

  The influence of iron nutrition on patterns of photosynthetic behavior, nitrogen metabolism, and fixed-carbon allocation is reported for a common freshwater green alga. Cultures of Scenedesmus quadricauda ...

  Citation × Citation Citation Abstract Copy Title: The role of iron nutrition in regulating patterns of photosynthesis and nitrogen metabolism in the green alga Scenedesmus quadricauda Author: Ades, Dennis Raymond Year: 1987 The influence of iron nutrition on patterns of photosynthetic behavior, nitrogen metabolism, and fixed-carbon allocation is reported for a common freshwater green alga. Cultures of Scenedesmus quadricauda were grown in Fraquil medium in which iron concentrations ranged from 1.0 μM to 0.01 μM (log 10-6 to 10-8 M, respectively). Carbon 14 and nitrogen uptake experiments were conducted at photosynthetically saturating and subsaturating photon flux densities. Title: The role of iron nutrition in regulating patterns of photosynthesis and nitrogen metabolism in the green alga Scenedesmus quadricauda Author: Ades, Dennis Raymond Year: 1987 The influence of iron nutrition on patterns of photosynthetic behavior, nitrogen metabolism, and fixed-carbon allocation is reported for a common freshwater green alga. Cultures of Scenedesmus quadricauda were grown in Fraquil medium in which iron concentrations ranged from 1.0 μM to 0.01 μM (log 10-6 to 10-8 M, respectively). Carbon 14 and nitrogen uptake experiments were conducted at photosynthetically saturating and subsaturating photon flux densities. Close

• 25. [Article] Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii

  Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and ...

  Citation × Citation Citation Abstract Copy Title: Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii Author: Warrier, Indu, Hicks, Linda D., Battisti, James M., Raghavan, Rahul, Minnick, Michael F. Year: 2014 Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated ($2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 59 UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription. Title: Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii Author: Warrier, Indu, Hicks, Linda D., Battisti, James M., Raghavan, Rahul, Minnick, Michael F. Year: 2014 Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated ($2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 59 UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription. Close

• 26. [Article] Transcriptional regulation of early progenitor competence in the Drosophila central nervous system

  xiii, 104 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.

  Citation × Citation Citation Abstract Copy Title: Transcriptional regulation of early progenitor competence in the Drosophila central nervous system Author: Tran, Khoa Dang, 1983- Year: 2011, 2010 xiii, 104 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. Title: Transcriptional regulation of early progenitor competence in the Drosophila central nervous system Author: Tran, Khoa Dang, 1983- Year: 2011, 2010 xiii, 104 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. Close

• 27. [Article] Why Do Animals Do What They Do, When They Do It? Characterizing the Role of the Hypothalamus-Pituitary-Adrenal Axis in Seasonal Life-History Transitions

  Resource availability follows seasonal cycles in environmental conditions. To align physiology and behavior with prevailing environmental conditions, seasonal animals integrate cues from the environment ...

  Citation × Citation Citation Abstract Copy Title: Why Do Animals Do What They Do, When They Do It? Characterizing the Role of the Hypothalamus-Pituitary-Adrenal Axis in Seasonal Life-History Transitions Author: Dayger Forbes, Catherine Anne Year: 2017 Resource availability follows seasonal cycles in environmental conditions. To align physiology and behavior with prevailing environmental conditions, seasonal animals integrate cues from the environment with their internal state. One of the systems animals use to integrate those cues is the hypothalamus-pituitary-adrenal (HPA) axis and its primary effector, glucocorticoid hormones. The HPA axis has wide-ranging effects on physiology and behavior and, in the context of a glucocorticoid stress response, is known to mediate tradeoffs between immediate survival and future fitness. The HPA axis also plays an important role in facilitating predictable life-history events. Variation in HPA axis activity has been reported in all vertebrates, often coordinating seasonal reproduction and possibly also transitions between life-history stages. My dissertation research used red-sided garter snakes (Thamnophis sirtalis parietalis) to examine the role of the HPA axis in regulating seasonal life-history transitions, especially in females In Chapter 2, I hypothesized that seasonal plasticity in stress responses is regulated, in part, by changes in the responsiveness of the adrenal glands to adrenocorticotropic hormone (ACTH). I found that glucocorticoid responses to ACTH challenge were smaller in males than in females during the spring, suggesting that reports of reduced stress responsiveness in males may reflect lower adrenal responsiveness to ACTH. The sex difference in mating season duration and consequently also in the timing of migration led me to hypothesize that sex differences in HPA axis activity could explain sex differences in the timing of migration. Furthermore, adrenal responsiveness to ACTH also varied seasonally in males, but not females, suggesting that female stress responses, which have not been studied, may not vary seasonally. In Chapter 3, I investigated potential seasonal variation in female stress responses, which have not previously been examined. In males, baseline glucocorticoids decrease over the course of the mating season resulting in significantly lower baseline levels in males that have begun to migrate. I hypothesized that a change in HPA axis activity occurs during spring and fall migration. Peak stress-induced glucocorticoid concentration occurred at an earlier sampling time in females during the spring compared to the fall. Peak stress-induced glucocorticoid concentrations also occurred at a later sampling time in migrating females than in pre-migratory females during the spring, suggesting that negative feedback regulation of the HPA axis changes as soon as females begin to migrate during the spring. Female red-sided garter snakes are biennial breeders that give birth approximately every other year implying that a female's recent reproductive history can influence whether or not she will reproduce in a given year. Body condition can be used as a proxy for recent reproductive history and can be related to baseline and stress-induced glucocorticoid concentrations. In Chapter 4, I hypothesized that hormonal and behavioral stress responses vary with body condition. Baseline glucocorticoids did not vary with body condition, but females in low body condition showed a significantly larger increase in plasma glucocorticoids in response to capture stress. Body condition, but not capture stress, influenced latency to copulate, suggesting that females are resistant to the behavioral effects of capture stress during the spring mating season. Only females in low body condition increased latency to copulate in response to injection of a physiological (15 µg) dose of exogenous CORT, while all females responded to a pharmacological (60 µg) dose, indicating that behavioral responses to exogenous glucocorticoids vary with female body condition. These data suggest that variation in body condition may be associated with differences in HPA axis sensitivity and/or glucocorticoid receptor (GR) density in the brain. I directly tested if there is a relationship among body condition, reproductive history and HPA axis activity in Chapter 5. I found that glucocorticoid stress responses and mating behavior did not vary with body condition, nor was body condition related to brain GR or reproductive condition (parturient vs post-parturient females). Only unreceptive females showed a significant stress-induced increase in glucocorticoids, suggesting that reduced stress responsiveness is associated with receptivity. Parturient females mated faster (were more proceptive) than post-parturient females. These data suggest that HPA axis activity influences reproductive "decisions" by modulating receptivity, while proceptivity is related primarily to recent reproductive history. Together, these chapters help characterize how HPA axis activity varies with season, sex, reproductive history and migration status. By systematically probing the HPA axis in a single, tractable system, I have gained insight into how changes in the HPA axis support and modulate transitions between life-history stages. These results highlight the HPA axis' important function in mediating the critical trade-offs all animals must navigate to be successful in a changing world. Title: Why Do Animals Do What They Do, When They Do It? Characterizing the Role of the Hypothalamus-Pituitary-Adrenal Axis in Seasonal Life-History Transitions Author: Dayger Forbes, Catherine Anne Year: 2017 Resource availability follows seasonal cycles in environmental conditions. To align physiology and behavior with prevailing environmental conditions, seasonal animals integrate cues from the environment with their internal state. One of the systems animals use to integrate those cues is the hypothalamus-pituitary-adrenal (HPA) axis and its primary effector, glucocorticoid hormones. The HPA axis has wide-ranging effects on physiology and behavior and, in the context of a glucocorticoid stress response, is known to mediate tradeoffs between immediate survival and future fitness. The HPA axis also plays an important role in facilitating predictable life-history events. Variation in HPA axis activity has been reported in all vertebrates, often coordinating seasonal reproduction and possibly also transitions between life-history stages. My dissertation research used red-sided garter snakes (Thamnophis sirtalis parietalis) to examine the role of the HPA axis in regulating seasonal life-history transitions, especially in females In Chapter 2, I hypothesized that seasonal plasticity in stress responses is regulated, in part, by changes in the responsiveness of the adrenal glands to adrenocorticotropic hormone (ACTH). I found that glucocorticoid responses to ACTH challenge were smaller in males than in females during the spring, suggesting that reports of reduced stress responsiveness in males may reflect lower adrenal responsiveness to ACTH. The sex difference in mating season duration and consequently also in the timing of migration led me to hypothesize that sex differences in HPA axis activity could explain sex differences in the timing of migration. Furthermore, adrenal responsiveness to ACTH also varied seasonally in males, but not females, suggesting that female stress responses, which have not been studied, may not vary seasonally. In Chapter 3, I investigated potential seasonal variation in female stress responses, which have not previously been examined. In males, baseline glucocorticoids decrease over the course of the mating season resulting in significantly lower baseline levels in males that have begun to migrate. I hypothesized that a change in HPA axis activity occurs during spring and fall migration. Peak stress-induced glucocorticoid concentration occurred at an earlier sampling time in females during the spring compared to the fall. Peak stress-induced glucocorticoid concentrations also occurred at a later sampling time in migrating females than in pre-migratory females during the spring, suggesting that negative feedback regulation of the HPA axis changes as soon as females begin to migrate during the spring. Female red-sided garter snakes are biennial breeders that give birth approximately every other year implying that a female's recent reproductive history can influence whether or not she will reproduce in a given year. Body condition can be used as a proxy for recent reproductive history and can be related to baseline and stress-induced glucocorticoid concentrations. In Chapter 4, I hypothesized that hormonal and behavioral stress responses vary with body condition. Baseline glucocorticoids did not vary with body condition, but females in low body condition showed a significantly larger increase in plasma glucocorticoids in response to capture stress. Body condition, but not capture stress, influenced latency to copulate, suggesting that females are resistant to the behavioral effects of capture stress during the spring mating season. Only females in low body condition increased latency to copulate in response to injection of a physiological (15 µg) dose of exogenous CORT, while all females responded to a pharmacological (60 µg) dose, indicating that behavioral responses to exogenous glucocorticoids vary with female body condition. These data suggest that variation in body condition may be associated with differences in HPA axis sensitivity and/or glucocorticoid receptor (GR) density in the brain. I directly tested if there is a relationship among body condition, reproductive history and HPA axis activity in Chapter 5. I found that glucocorticoid stress responses and mating behavior did not vary with body condition, nor was body condition related to brain GR or reproductive condition (parturient vs post-parturient females). Only unreceptive females showed a significant stress-induced increase in glucocorticoids, suggesting that reduced stress responsiveness is associated with receptivity. Parturient females mated faster (were more proceptive) than post-parturient females. These data suggest that HPA axis activity influences reproductive "decisions" by modulating receptivity, while proceptivity is related primarily to recent reproductive history. Together, these chapters help characterize how HPA axis activity varies with season, sex, reproductive history and migration status. By systematically probing the HPA axis in a single, tractable system, I have gained insight into how changes in the HPA axis support and modulate transitions between life-history stages. These results highlight the HPA axis' important function in mediating the critical trade-offs all animals must navigate to be successful in a changing world. Close

• 28. [Article] Androgen-induced immunosuppression

  It is well established that females are more immunocompetent than males as evidenced by higher humoral antibody titers, lowered susceptibility to infection, and more efficient graft rejection. Furthermore, ...

  Citation × Citation Citation Abstract Copy Title: Androgen-induced immunosuppression Author: Weyant, Debra Ann Year: 1979 It is well established that females are more immunocompetent than males as evidenced by higher humoral antibody titers, lowered susceptibility to infection, and more efficient graft rejection. Furthermore, females also exhibit a much higher incidence of autoimmune disease. These observations have led investigators to believe that the male hormonal environment may play a key role in the regulation of immune response. For this reason, this study is concerned with the expression of autoimmunity and of immune function in the mouse. This study included the New Zealand Black (NZB) mouse strain, as an animal model for human SLE, as well as normal DBA/2 and Balb/c strains. Animals were administered testosterone via subcutaneous implants in silastic tubing or by injection. Mice used were intact females, intact males and castrated males. Animals were otherwise untreated or had been exposed to a sublethal dose (400-550 rads) of irradiation. Target organ weight changes, immune capacity and peripheral blood picture changes were measured. Title: Androgen-induced immunosuppression Author: Weyant, Debra Ann Year: 1979 It is well established that females are more immunocompetent than males as evidenced by higher humoral antibody titers, lowered susceptibility to infection, and more efficient graft rejection. Furthermore, females also exhibit a much higher incidence of autoimmune disease. These observations have led investigators to believe that the male hormonal environment may play a key role in the regulation of immune response. For this reason, this study is concerned with the expression of autoimmunity and of immune function in the mouse. This study included the New Zealand Black (NZB) mouse strain, as an animal model for human SLE, as well as normal DBA/2 and Balb/c strains. Animals were administered testosterone via subcutaneous implants in silastic tubing or by injection. Mice used were intact females, intact males and castrated males. Animals were otherwise untreated or had been exposed to a sublethal dose (400-550 rads) of irradiation. Target organ weight changes, immune capacity and peripheral blood picture changes were measured. Close

• 29. [Article] Decrease in selected temperature after intracranial dopamine injections in goldfish

  Goldfish (Carassius auratus) (40-80g) were injected with dopamine into the forebrain to study the possible involvement of this amine in central temperature regulation in these fish. Dopamine caused a decrease ...

  Citation × Citation Citation Abstract Copy Title: Decrease in selected temperature after intracranial dopamine injections in goldfish Author: Panayiotides-Djaferis, Hercules Theodore Year: 1987 Goldfish (Carassius auratus) (40-80g) were injected with dopamine into the forebrain to study the possible involvement of this amine in central temperature regulation in these fish. Dopamine caused a decrease in selected temperature after injection into the rostral nucleus preopticus periventricularis (NPP). This effect was dependent on the dose of dopamine administered. Doses of 25, 50, 100 and 250 ng were used, injected in a volume of 0.2μ1. Injections in regions adjacent to the NPP elicited hypothermic effects only at the higher dosages. These effects were not consistent. Injections in caudal regions of the NPP elicited no effect. The effects of dopamine were blocked by haloperidol, a selective antagonist of dopamine. It is suggested that dopamine acts on central thermoregulatory neurons, present in the rostral NPP, in the mediation of thermoregulatory behavior. Further, it is suggested that this action is mediated via dopaminergic receptors. Title: Decrease in selected temperature after intracranial dopamine injections in goldfish Author: Panayiotides-Djaferis, Hercules Theodore Year: 1987 Goldfish (Carassius auratus) (40-80g) were injected with dopamine into the forebrain to study the possible involvement of this amine in central temperature regulation in these fish. Dopamine caused a decrease in selected temperature after injection into the rostral nucleus preopticus periventricularis (NPP). This effect was dependent on the dose of dopamine administered. Doses of 25, 50, 100 and 250 ng were used, injected in a volume of 0.2μ1. Injections in regions adjacent to the NPP elicited hypothermic effects only at the higher dosages. These effects were not consistent. Injections in caudal regions of the NPP elicited no effect. The effects of dopamine were blocked by haloperidol, a selective antagonist of dopamine. It is suggested that dopamine acts on central thermoregulatory neurons, present in the rostral NPP, in the mediation of thermoregulatory behavior. Further, it is suggested that this action is mediated via dopaminergic receptors. Close

• 30. [Article] A Genetic Study of SSV1, the Prototypical Fusellovirus

  Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology ...

  Citation × Citation Citation Abstract Copy Title: A Genetic Study of SSV1, the Prototypical Fusellovirus Author: Iverson, Eric, Stedman, Kenneth M. Year: 2012 Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu, Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly conserved predicted DNA-binding protein gene VP2 yields viable virus that is indistinguishable from wild-type. Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However, infection of S. solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus. Title: A Genetic Study of SSV1, the Prototypical Fusellovirus Author: Iverson, Eric, Stedman, Kenneth M. Year: 2012 Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu, Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly conserved predicted DNA-binding protein gene VP2 yields viable virus that is indistinguishable from wild-type. Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However, infection of S. solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus. Close

• 31. [Article] Norepinephrine and temperature regulation in goldfish

  Cannulae were implanted into forebrain loci of goldfish (Carassius auratus; 45-90 g) to determine (i) the effects and site of action of intracranial norepinephrine (NE) injections on behavioral thermoregulation ...

  Citation × Citation Citation Abstract Copy Title: Norepinephrine and temperature regulation in goldfish Author: Wollmuth, Lonnie Paul Year: 1987 Cannulae were implanted into forebrain loci of goldfish (Carassius auratus; 45-90 g) to determine (i) the effects and site of action of intracranial norepinephrine (NE) injections on behavioral thermoregulation and (ii) the mechanism and the types of adrenoreceptors involved in the thermoregulatory effect of NE. After 30 min in a thermal gradient, implanted fish were injected with norepinephrinebitartrate salt (2.5-500 ng NE) in a total volume of 0.2 ul (carrier was 0.7% NaCl). Injections of 5, 10, 25, and 50 ng NE into the anterior aspect of the nucleus preopticus periventricularis (NPP1 Peter and Gill 1975) led to consistent, dose-dependent decreases in selected temperature. No effect on temperature selection was observed following injections of 2.5 ng NE or control injections of 100 ng tartaric acid. The effects of injections into other loci, including intraventricular injections, were dependent upon the dose and proximity to the anterior NPP1 at sites adjacent to the anterior NPP, larger doses were required, and the effects became inconsistent. At sites further removed, no effect on selected temperature was observed1 included in this category were more caudal sites within the NPP and the nucleus preopticus. Title: Norepinephrine and temperature regulation in goldfish Author: Wollmuth, Lonnie Paul Year: 1987 Cannulae were implanted into forebrain loci of goldfish (Carassius auratus; 45-90 g) to determine (i) the effects and site of action of intracranial norepinephrine (NE) injections on behavioral thermoregulation and (ii) the mechanism and the types of adrenoreceptors involved in the thermoregulatory effect of NE. After 30 min in a thermal gradient, implanted fish were injected with norepinephrinebitartrate salt (2.5-500 ng NE) in a total volume of 0.2 ul (carrier was 0.7% NaCl). Injections of 5, 10, 25, and 50 ng NE into the anterior aspect of the nucleus preopticus periventricularis (NPP1 Peter and Gill 1975) led to consistent, dose-dependent decreases in selected temperature. No effect on temperature selection was observed following injections of 2.5 ng NE or control injections of 100 ng tartaric acid. The effects of injections into other loci, including intraventricular injections, were dependent upon the dose and proximity to the anterior NPP1 at sites adjacent to the anterior NPP, larger doses were required, and the effects became inconsistent. At sites further removed, no effect on selected temperature was observed1 included in this category were more caudal sites within the NPP and the nucleus preopticus. Close

• 32. [Article] Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus

  This is chapter 1 from Anoxia -- This book reviews how severe oxygen deprivation affects biological systems - from the molecular to the ecological level. The contributing authors come from diverse regions ...

  Citation × Citation Citation Abstract Copy Title: Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus Author: Podrabsky, Jason E., Riggs, Claire L., Duerr, Jeffrey M. Year: 2012 This is chapter 1 from Anoxia -- This book reviews how severe oxygen deprivation affects biological systems - from the molecular to the ecological level. The contributing authors come from diverse regions of the world, which proves the interest in the academic analysis of oxygen deprivation. The diversity in the experimental approach scientists take, in order to understand the influence oxygen deprivation has on living systems, is apparent throughout this book. One of the presented ideas deals with the exploration and examination of the physiological, cellular and genetic characteristics of killifish embryos and nematodes exposed to anoxia. Furthermore, the book includes material on the mechanisms regulating hypoxia and anoxia tolerance and their implications of on human health issues. Finally, new methodologies to examine oxygen deprivation and the impact of human-related activities on oxygen level, within important ecological systems such as Lake Victoria, are presented. There is no doubt that the oxygen molecule is central to every stratum of biological systems. Title: Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus Author: Podrabsky, Jason E., Riggs, Claire L., Duerr, Jeffrey M. Year: 2012 This is chapter 1 from Anoxia -- This book reviews how severe oxygen deprivation affects biological systems - from the molecular to the ecological level. The contributing authors come from diverse regions of the world, which proves the interest in the academic analysis of oxygen deprivation. The diversity in the experimental approach scientists take, in order to understand the influence oxygen deprivation has on living systems, is apparent throughout this book. One of the presented ideas deals with the exploration and examination of the physiological, cellular and genetic characteristics of killifish embryos and nematodes exposed to anoxia. Furthermore, the book includes material on the mechanisms regulating hypoxia and anoxia tolerance and their implications of on human health issues. Finally, new methodologies to examine oxygen deprivation and the impact of human-related activities on oxygen level, within important ecological systems such as Lake Victoria, are presented. There is no doubt that the oxygen molecule is central to every stratum of biological systems. Close

• 33. [Article] Nitrogen and iron interactions in filamentous cyanobacteria

  The investigations described in this paper are an attempt to further define and quantify the interrelationship of nitrogen fixation and iron nutritional physiology in these two species. Chapter II will ...

  Citation × Citation Citation Abstract Copy Title: Nitrogen and iron interactions in filamentous cyanobacteria Author: Hutchins, David Allen Year: 1989 The investigations described in this paper are an attempt to further define and quantify the interrelationship of nitrogen fixation and iron nutritional physiology in these two species. Chapter II will present and compare data on nutritional ratios of field collected Trichodesmium colonies and laboratory Anabaena cultures, with the intent of examining possible correlations between observed iron levels and protein nitrogen and chlorophyll concentrations, as well as nitrogen fixation rates. Chapter Ill is an examination of nitrogen fixation and siderophore production in Anabaena with emphasis on the possible implications of hypothesized synergistic effects of these two physiological capabilities on cyanobacterial dominance and bloom formation. Chapter IV will deal with the possibility of gratuitous manganese repression of Anabaena siderophore production in the manner described by Hantke (1987) for regulation of siderophore production in E. coli. The Conclusions chapter (Chapter V) will present a discussion of the results of these experiments in the context of current problems in cyanobacterial physiology, ecology and evolution. It is hoped that a significant contribution can be made to our understanding of the related problems of cyanobacterial dominance in freshwater ecosysytems and the scarcity of cyanobacterial nitrogen fixation in marine ecosystems. Title: Nitrogen and iron interactions in filamentous cyanobacteria Author: Hutchins, David Allen Year: 1989 The investigations described in this paper are an attempt to further define and quantify the interrelationship of nitrogen fixation and iron nutritional physiology in these two species. Chapter II will present and compare data on nutritional ratios of field collected Trichodesmium colonies and laboratory Anabaena cultures, with the intent of examining possible correlations between observed iron levels and protein nitrogen and chlorophyll concentrations, as well as nitrogen fixation rates. Chapter Ill is an examination of nitrogen fixation and siderophore production in Anabaena with emphasis on the possible implications of hypothesized synergistic effects of these two physiological capabilities on cyanobacterial dominance and bloom formation. Chapter IV will deal with the possibility of gratuitous manganese repression of Anabaena siderophore production in the manner described by Hantke (1987) for regulation of siderophore production in E. coli. The Conclusions chapter (Chapter V) will present a discussion of the results of these experiments in the context of current problems in cyanobacterial physiology, ecology and evolution. It is hoped that a significant contribution can be made to our understanding of the related problems of cyanobacterial dominance in freshwater ecosysytems and the scarcity of cyanobacterial nitrogen fixation in marine ecosystems. Close

• 34. [Article] Emergence of New sRNAs in Enteric Bacteria is Associated with Low Expression and Rapid Evolution

  Non-coding small RNAs (sRNAs) are critical to post-transcriptional gene regulation in bacteria. However, unlike for protein-coding genes, the evolutionary forces that shape sRNAs are not understood. We ...

  Citation × Citation Citation Abstract Copy Title: Emergence of New sRNAs in Enteric Bacteria is Associated with Low Expression and Rapid Evolution Author: Kacharia, Fenil R., Millar, Jess A., Raghavan, Rahul Year: 2017 Non-coding small RNAs (sRNAs) are critical to post-transcriptional gene regulation in bacteria. However, unlike for protein-coding genes, the evolutionary forces that shape sRNAs are not understood. We investigated sRNAs in enteric bacteria and discovered that recently emerged sRNAs evolve at significantly faster rates than older sRNAs. Concomitantly, younger sRNAs are expressed at significantly lower levels than older sRNAs. This process could potentially facilitate the integration of newly emerged sRNAs into bacterial regulatory networks. Furthermore, it has previously been difficult to trace the evolutionary histories of sRNAs because rapid evolution obscures their original sources. We overcame this challenge by identifying a recently evolved sRNA in Escherichia coli, which allowed us to determine that novel sRNAs could emerge from vestigial bacteriophage genes, the first known source for sRNA origination. Title: Emergence of New sRNAs in Enteric Bacteria is Associated with Low Expression and Rapid Evolution Author: Kacharia, Fenil R., Millar, Jess A., Raghavan, Rahul Year: 2017 Non-coding small RNAs (sRNAs) are critical to post-transcriptional gene regulation in bacteria. However, unlike for protein-coding genes, the evolutionary forces that shape sRNAs are not understood. We investigated sRNAs in enteric bacteria and discovered that recently emerged sRNAs evolve at significantly faster rates than older sRNAs. Concomitantly, younger sRNAs are expressed at significantly lower levels than older sRNAs. This process could potentially facilitate the integration of newly emerged sRNAs into bacterial regulatory networks. Furthermore, it has previously been difficult to trace the evolutionary histories of sRNAs because rapid evolution obscures their original sources. We overcame this challenge by identifying a recently evolved sRNA in Escherichia coli, which allowed us to determine that novel sRNAs could emerge from vestigial bacteriophage genes, the first known source for sRNA origination. Close

• 35. [Article] Analyzing Early Embryonic Cell Division Defects in Caenorhabditis Elegans

  36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2...

  Citation × Citation Citation Abstract Copy Title: Analyzing Early Embryonic Cell Division Defects in Caenorhabditis Elegans Author: Krishnakumar, Sheetal Year: 2014 36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2014. Title: Analyzing Early Embryonic Cell Division Defects in Caenorhabditis Elegans Author: Krishnakumar, Sheetal Year: 2014 36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2014. Close

• 36. [Article] The Response of Mice to Infection by the Parasitic Nematode Trichinella: A Comparison of Trichinella Spiralis and Trichinella Pseudospiralis

  The intracellular parasite Trichinella is a genus in phylum Nematoda that contains six named species including Trichinella spiralis and Trichinella pseudospiralis. These parasites infect a large variety ...

  Citation × Citation Citation Abstract Copy Title: The Response of Mice to Infection by the Parasitic Nematode Trichinella: A Comparison of Trichinella Spiralis and Trichinella Pseudospiralis Author: Anderson, Barry Clayton Year: 2002 The intracellular parasite Trichinella is a genus in phylum Nematoda that contains six named species including Trichinella spiralis and Trichinella pseudospiralis. These parasites infect a large variety of wild and domestic animals, human beings and a few species of birds. The parasitic strategies and the pathological effects on the host between trichinella spiralis and Trichinella pseudospiralis are quite different. The purpose of this study is to gain an understanding of the physiological, immunological and pathological differences between these two species of Trichinella using infections in the mouse as a model. In the course of this research I have attempted to answer the following questions: A) Is cortisol a factor in the differences of the host response to Trichinella spiralis or Trichinella pseudospiralis? B) Are there differences in leukocyte response in the peripheral blood of Trichinella infected mice? C) Are there differences in the up-regulation or down-regulation of cell surface molecules on leukocytes in the spleens of Trichinella infected mice? D) Is there a difference in the degree of muscle damage (as measured by creatine phosphokinase) when infections by the two species of Trichinella are compared? E) Are there differences in angiogenesis and collagen deposition in Trichinella infected mice and are these differences related to cortisol? F) Is nitric oxide a component in parasite killing and are there differences in nitric oxide production in host mice when the two species of Trichinella are compared? My research has shown that there are significant differences in the parasitic strategies and pathological consequences in mice infected with one or the other of the two species of Trichinella. The two species appear to generate different immune and inflammatory responses from the host. Trichinella pseudospiralis is much less damaging to the host, generates a very different peripheral blood response, stimulates the production of substantially greater levels of serum cortisol, generates a significantly different profile in cytokine production presents a very different cell surface antigen profile and does not produce a collagen nurse cell or generate an angiogenic response when compared to T spiralis. In addition, I have shown a role for nitric oxide in parasite killing and a role for serum cortisol in larval survival. I have also shown that cortisol has no role in either collagen deposition or the angiogenic response in Balb/c mice under the experimental conditions detailed here. Title: The Response of Mice to Infection by the Parasitic Nematode Trichinella: A Comparison of Trichinella Spiralis and Trichinella Pseudospiralis Author: Anderson, Barry Clayton Year: 2002 The intracellular parasite Trichinella is a genus in phylum Nematoda that contains six named species including Trichinella spiralis and Trichinella pseudospiralis. These parasites infect a large variety of wild and domestic animals, human beings and a few species of birds. The parasitic strategies and the pathological effects on the host between trichinella spiralis and Trichinella pseudospiralis are quite different. The purpose of this study is to gain an understanding of the physiological, immunological and pathological differences between these two species of Trichinella using infections in the mouse as a model. In the course of this research I have attempted to answer the following questions: A) Is cortisol a factor in the differences of the host response to Trichinella spiralis or Trichinella pseudospiralis? B) Are there differences in leukocyte response in the peripheral blood of Trichinella infected mice? C) Are there differences in the up-regulation or down-regulation of cell surface molecules on leukocytes in the spleens of Trichinella infected mice? D) Is there a difference in the degree of muscle damage (as measured by creatine phosphokinase) when infections by the two species of Trichinella are compared? E) Are there differences in angiogenesis and collagen deposition in Trichinella infected mice and are these differences related to cortisol? F) Is nitric oxide a component in parasite killing and are there differences in nitric oxide production in host mice when the two species of Trichinella are compared? My research has shown that there are significant differences in the parasitic strategies and pathological consequences in mice infected with one or the other of the two species of Trichinella. The two species appear to generate different immune and inflammatory responses from the host. Trichinella pseudospiralis is much less damaging to the host, generates a very different peripheral blood response, stimulates the production of substantially greater levels of serum cortisol, generates a significantly different profile in cytokine production presents a very different cell surface antigen profile and does not produce a collagen nurse cell or generate an angiogenic response when compared to T spiralis. In addition, I have shown a role for nitric oxide in parasite killing and a role for serum cortisol in larval survival. I have also shown that cortisol has no role in either collagen deposition or the angiogenic response in Balb/c mice under the experimental conditions detailed here. Close

• 37. [Article] Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice

  The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated ...

  Citation × Citation Citation Abstract Copy Title: Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice Author: Claiborn, Kathryn C., Sachdeva, Mira M., Cannon, Corey E., Groff, David N., Singer, Jeffrey D., Stoffers, Doris A. Year: 2010 The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis. Title: Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice Author: Claiborn, Kathryn C., Sachdeva, Mira M., Cannon, Corey E., Groff, David N., Singer, Jeffrey D., Stoffers, Doris A. Year: 2010 The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis. Close

• 38. [Article] The Hyperthermophilic Archaeon Sulfolobus : from Exploration to Exploitation

  In the early 1970s, Sulfolobus was first isolated by Thomas Brock and co-workers from sulfur-rich acidic hot springs in Yellowstone National Park. Sulfolobus became one of the model organisms of Archaea ...

  Citation × Citation Citation Abstract Copy Title: The Hyperthermophilic Archaeon Sulfolobus : from Exploration to Exploitation Author: Brouns, Stan J.J., Ettema, Thijs J.G., Stedman, Kenneth M., Walther, Jasper, Smith, Hauke, Snijders, Ambrosius P.L., Young, Mark, Bernander, Rolf, Wright, Phillip C., Siebers, Betina, Van der Oost, John Year: 2003 In the early 1970s, Sulfolobus was first isolated by Thomas Brock and co-workers from sulfur-rich acidic hot springs in Yellowstone National Park. Sulfolobus became one of the model organisms of Archaea in general, and of Crenarchaea in particular. Many of its unusual physiological characteristics have been investigated, and several of its thermostable enzymes have been studied in considerable detail. For fundamental reasons, and because of its potential for industrial applications, Sulfolobus has been selected for a genome sequence project. The recent completion of the Sulfolobus solfataricus genome has set the stage for a series of postgenome research lines that will be reviewed here. Comparative genomics aims to unravel the cell’s metabolic potential (enzymes, pathways, and regulation) and compare it to other organisms. This in silico analysis of Sulfolobus has revealed several unique metabolic features and confirmed that the major control processes (transcription, translation, and replication) resemble eukaryal much more than bacterial equivalents. Currently, several research groups use a functional genomics approach—a genomebased, high-throughput analysis of the complete Sulfolobus cell—at the level of RNA (transcriptomics), protein (proteomics), and metabolic intermediates and products (metabolomics). Apart from analyzing the cell’s response to different cultivation conditions, the comparison of different genotypes (wild type and mutants) would be very interesting for both basic science and applied reasons. Much effort has recently been put into the establishment of tools that allow genetic engineering of Sulfolobus. The future challenge is to integrate available knowledge in order to understand the relevant mechanisms that enable Sulfolobus to thrive in its extreme habitats. Moreover, such a Systems Biology approach is essential as a basis for the directed engineering and industrial exploitation of this unique microorganism. Title: The Hyperthermophilic Archaeon Sulfolobus : from Exploration to Exploitation Author: Brouns, Stan J.J., Ettema, Thijs J.G., Stedman, Kenneth M., Walther, Jasper, Smith, Hauke, Snijders, Ambrosius P.L., Young, Mark, Bernander, Rolf, Wright, Phillip C., Siebers, Betina, Van der Oost, John Year: 2003 In the early 1970s, Sulfolobus was first isolated by Thomas Brock and co-workers from sulfur-rich acidic hot springs in Yellowstone National Park. Sulfolobus became one of the model organisms of Archaea in general, and of Crenarchaea in particular. Many of its unusual physiological characteristics have been investigated, and several of its thermostable enzymes have been studied in considerable detail. For fundamental reasons, and because of its potential for industrial applications, Sulfolobus has been selected for a genome sequence project. The recent completion of the Sulfolobus solfataricus genome has set the stage for a series of postgenome research lines that will be reviewed here. Comparative genomics aims to unravel the cell’s metabolic potential (enzymes, pathways, and regulation) and compare it to other organisms. This in silico analysis of Sulfolobus has revealed several unique metabolic features and confirmed that the major control processes (transcription, translation, and replication) resemble eukaryal much more than bacterial equivalents. Currently, several research groups use a functional genomics approach—a genomebased, high-throughput analysis of the complete Sulfolobus cell—at the level of RNA (transcriptomics), protein (proteomics), and metabolic intermediates and products (metabolomics). Apart from analyzing the cell’s response to different cultivation conditions, the comparison of different genotypes (wild type and mutants) would be very interesting for both basic science and applied reasons. Much effort has recently been put into the establishment of tools that allow genetic engineering of Sulfolobus. The future challenge is to integrate available knowledge in order to understand the relevant mechanisms that enable Sulfolobus to thrive in its extreme habitats. Moreover, such a Systems Biology approach is essential as a basis for the directed engineering and industrial exploitation of this unique microorganism. Close

• 39. [Article] Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells

  Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it ...

  Citation × Citation Citation Abstract Copy Title: Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells Author: Singer, Jeffrey D., Gurian-West, Mark, Clurman, Bruce, Roberts, James M. Year: 1999 Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin–protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance. Title: Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells Author: Singer, Jeffrey D., Gurian-West, Mark, Clurman, Bruce, Roberts, James M. Year: 1999 Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin–protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance. Close

• 40. [Article] Microbial Biomineralization of Iron

  Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented ...

  Citation × Citation Citation Abstract Copy Title: Microbial Biomineralization of Iron Author: Fang, Wen Year: 2013 Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented in this thesis focused on two types of microorganisms capable of forming iron biominerals. In the first project I have studied the effect of O2 on the respiratory physiology and the formation of magnetosomes by Magnetospirillum magneticum AMB-1. In the second project I have studied the relationship between olivine and the activity of dissimilatory sulfate reducing (DSR) microorganisms. For the first project, I grew cells of AMB-1 in cultures with various concentrations of O2 and monitored growth and the formation of magnetic mineral particles (MMP). Results have shown that AMB-1 cells grew better at 100-225 uMO2(aq) than at lower [O2], yet the formation of MMP was repressed at ~45 uM O2(aq) and strongly inhibited at >100 uM O2(aq).These results have helped better understand the dissimilarity between the optimal growth conditions of magnetotactic bacteria and the conditions needed for the formation of MMPs. My results have also shown that the reaction between H2S produced by DSRs and olivine is abiotic, not catalyzed and exergonic. The pH did not vary significantly during this reaction and pH variation (in the 5-9 range) did not significantly influence this chemical reaction. Bicarbonate inhibited the reaction between H2S and olivine, but not the chemical equilibrium. Phosphate, a weak iron chelator, influenced the equilibrium of the reaction and it is assumed to help increase the rate of olivine weathering in the presence of DSRs. The activity of DSRs was positively influenced by the presence and abundance of olivine. Based on my results I propose that olivine help DSR obtain energy more efficiently, but does not represent a source of energy or nutrients for the cells. These results helped better understand the formation of iron biominerals and signatures of this activity. Title: Microbial Biomineralization of Iron Author: Fang, Wen Year: 2013 Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented in this thesis focused on two types of microorganisms capable of forming iron biominerals. In the first project I have studied the effect of O2 on the respiratory physiology and the formation of magnetosomes by Magnetospirillum magneticum AMB-1. In the second project I have studied the relationship between olivine and the activity of dissimilatory sulfate reducing (DSR) microorganisms. For the first project, I grew cells of AMB-1 in cultures with various concentrations of O2 and monitored growth and the formation of magnetic mineral particles (MMP). Results have shown that AMB-1 cells grew better at 100-225 uMO2(aq) than at lower [O2], yet the formation of MMP was repressed at ~45 uM O2(aq) and strongly inhibited at >100 uM O2(aq).These results have helped better understand the dissimilarity between the optimal growth conditions of magnetotactic bacteria and the conditions needed for the formation of MMPs. My results have also shown that the reaction between H2S produced by DSRs and olivine is abiotic, not catalyzed and exergonic. The pH did not vary significantly during this reaction and pH variation (in the 5-9 range) did not significantly influence this chemical reaction. Bicarbonate inhibited the reaction between H2S and olivine, but not the chemical equilibrium. Phosphate, a weak iron chelator, influenced the equilibrium of the reaction and it is assumed to help increase the rate of olivine weathering in the presence of DSRs. The activity of DSRs was positively influenced by the presence and abundance of olivine. Based on my results I propose that olivine help DSR obtain energy more efficiently, but does not represent a source of energy or nutrients for the cells. These results helped better understand the formation of iron biominerals and signatures of this activity. Close

• 41. [Article] Patterns of Stress Responses Shift During Seasonal Life-History Transitions: An Analysis Comparing Baseline, Maximal and Integrated Corticosterone in Female Red-Sided Garter Snakes (Thamnophis sirtalis parietalis)

  Glucocorticoids often rise and fall with a variety of external and internal cues and frequently vary among life-history stages. This suggests that changing glucocorticoids may coordinate life-history transitions. ...

  Citation × Citation Citation Abstract Copy Title: Patterns of Stress Responses Shift During Seasonal Life-History Transitions: An Analysis Comparing Baseline, Maximal and Integrated Corticosterone in Female Red-Sided Garter Snakes (Thamnophis sirtalis parietalis) Author: Dayger, Catherine A., Lutterschmidt, Deborah I. Year: 2017 Glucocorticoids often rise and fall with a variety of external and internal cues and frequently vary among life-history stages. This suggests that changing glucocorticoids may coordinate life-history transitions. To explore this hypothesis, we asked if the time-course of stress-induced glucocorticoid levels differ between two life-history transitions (i.e., spring and fall migration) in female red-sided garter snakes (Thamnophis sirtalis parietalis). We collected non-migratory females from a communal den and migratory females from a road along the migration route and treated them with 4 h of capture stress; plasma corticosterone was measured before, during and after capture stress. During the spring, den-collected females exhibited a stress-induced peak in corticosterone at an earlier sampling time than migrating, road-collected females. Because the pattern of corticosterone responses varied with migratory state, negative feedback on and/or sensitivity of the hypothalamus-pituitaryadrenal (HPA) axis may be linked to spring migration. During the fall, capture stress elicited an increase in corticosterone in den-collected females but not in migrating, road-collected females. Baseline corticosterone was higher and both maximal and integrated corticosterone responses were lower during the fall compared to spring, indicating that stress responses are smaller when baseline corticosterone is elevated, perhaps due to a “ceiling effect”. These data suggest that HPA axis regulation changes during seasonal migration, possibly via altering negative feedback, HPA axis sensitivity, or some other mechanism. This study supports the hypothesis that glucocorticoids coordinate life-history events and suggests that examining a suite of stress response characteristics is most informative for understanding the function of HPA modulation. Title: Patterns of Stress Responses Shift During Seasonal Life-History Transitions: An Analysis Comparing Baseline, Maximal and Integrated Corticosterone in Female Red-Sided Garter Snakes (Thamnophis sirtalis parietalis) Author: Dayger, Catherine A., Lutterschmidt, Deborah I. Year: 2017 Glucocorticoids often rise and fall with a variety of external and internal cues and frequently vary among life-history stages. This suggests that changing glucocorticoids may coordinate life-history transitions. To explore this hypothesis, we asked if the time-course of stress-induced glucocorticoid levels differ between two life-history transitions (i.e., spring and fall migration) in female red-sided garter snakes (Thamnophis sirtalis parietalis). We collected non-migratory females from a communal den and migratory females from a road along the migration route and treated them with 4 h of capture stress; plasma corticosterone was measured before, during and after capture stress. During the spring, den-collected females exhibited a stress-induced peak in corticosterone at an earlier sampling time than migrating, road-collected females. Because the pattern of corticosterone responses varied with migratory state, negative feedback on and/or sensitivity of the hypothalamus-pituitaryadrenal (HPA) axis may be linked to spring migration. During the fall, capture stress elicited an increase in corticosterone in den-collected females but not in migrating, road-collected females. Baseline corticosterone was higher and both maximal and integrated corticosterone responses were lower during the fall compared to spring, indicating that stress responses are smaller when baseline corticosterone is elevated, perhaps due to a “ceiling effect”. These data suggest that HPA axis regulation changes during seasonal migration, possibly via altering negative feedback, HPA axis sensitivity, or some other mechanism. This study supports the hypothesis that glucocorticoids coordinate life-history events and suggests that examining a suite of stress response characteristics is most informative for understanding the function of HPA modulation. Close

• 42. [Article] Genome Rearrangements Can Make and Break Small RNA Genes

  Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are ...

  Citation × Citation Citation Abstract Copy Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Title: Genome Rearrangements Can Make and Break Small RNA Genes Author: Raghavan, Rahul, Kacharia, Fenil R., Millar, Jess A, Sislak, Christine Demko, Ochman, Howard Year: 2015 Small RNAs (sRNAs) are short, transcribed regulatory elements that are typically encoded in the intergenic regions (IGRs) of bacterial genomes. Several sRNAs, first recognized in Escherichia coli, are conserved among enteric bacteria, but because of the regulatory roles of sRNAs, differences in sRNA repertoires might be responsible for features that differentiate closely related species. We scanned the E. coli MG1655 and Salmonella enterica Typhimurium genomes for nonsyntenic IGRs as a potential source of uncharacterized, species-specific sRNAs and found that genome rearrangements have reconfigured several IGRs causing the disruption and formation of sRNAs. Within an IGR that is present in E. coli but was disrupted in Salmonella by a translocation event is an sRNA that is associated with the FNR/CRP global regulators and influences E. coli biofilm formation. A Salmonella-specific sRNA evolved de novo through point mutations that generated a σ70 promoter sequence in an IGR that arose through genome rearrangement events. The differences in the sRNA pools among bacterial species have previously been ascribed to duplication, deletion, or horizontal acquisition. Here, we show that genomic rearrangements also contribute to this process by either disrupting sRNA-containing IGRs or creating IGRs in which novel sRNAs may evolve. Close

• 43. [Article] Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos

  Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially ...

  Citation × Citation Citation Abstract Copy Title: Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance. Title: Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance. Close

• 44. [Article] Characterization of Functional Domains of Cul3, an E3 Ubiquitin Ligase, Using Chimeric Analysis

  Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in ...

  Citation × Citation Citation Abstract Copy Title: Characterization of Functional Domains of Cul3, an E3 Ubiquitin Ligase, Using Chimeric Analysis Author: Mitchell, Jennifer Anne Year: 2014 Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells. In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells. The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments. In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function. Title: Characterization of Functional Domains of Cul3, an E3 Ubiquitin Ligase, Using Chimeric Analysis Author: Mitchell, Jennifer Anne Year: 2014 Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells. In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells. The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments. In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function. Close

• 45. [Article] Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i>

  In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. ...

  Citation × Citation Citation Abstract Copy Title: Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Wagner, Josiah Tad Year: 2015 In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause. Title: Developmental Mechanisms that Support Genome Stability and Embryonic Survival in Stress-tolerant Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Wagner, Josiah Tad Year: 2015 In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause. Close

• 46. [Article] Antisense transcription is pervasive but rarely conserved in enteric bacteria

  Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent ...

  Citation × Citation Citation Abstract Copy Title: Antisense transcription is pervasive but rarely conserved in enteric bacteria Author: Raghavan, Rahul, Sloan, D. B., Ochman, H. Year: 2012 Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional. Title: Antisense transcription is pervasive but rarely conserved in enteric bacteria Author: Raghavan, Rahul, Sloan, D. B., Ochman, H. Year: 2012 Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional. Close

• 47. [Article] Predicting Parturition in a Long-Gestating Species: Behavioral and Hormonal Indicators in the Asian Elephant (Elephas maximus)

  Captive populations of Asian elephants (Elephas maximus) in North America are not self-sustaining, and increasing reproductive success within captive populations is a high priority. The ability to accurately ...

  Citation × Citation Citation Abstract Copy Title: Predicting Parturition in a Long-Gestating Species: Behavioral and Hormonal Indicators in the Asian Elephant (Elephas maximus) Author: Velonis, Heather Kelly Year: 2017 Captive populations of Asian elephants (Elephas maximus) in North America are not self-sustaining, and increasing reproductive success within captive populations is a high priority. The ability to accurately predict parturition can have a direct impact on elephant welfare. Elephants in captivity often require significant preparation and management throughout the birthing process, and complications during labor and delivery can necessitate immediate intervention, including stillbirth, protracted labor, maternal aggression towards a newborn calf, and dystocia. Being able to predict when parturition will commence can ensure appropriate staff is available and adequate monitoring is performed. Routine endocrine sampling can be used to predict parturition in Asian elephants, with a drop in progesterone (P4) to baseline levels signaling parturition in 2-5 days. However, we determined this method is not without limitations, and it is not used in all institutions that house elephants. As changes in hormones regulate and alter behaviors, we investigated behavioral indicators as an additional management tool for predicting parturition, a time of drastic hormone changes. We conducted a study of five pregnancies in Asian elephants at the Oregon Zoo, U.S.A, and Taronga Zoo, Australia, between 2008 and 2012. In Chapter 2, I evaluated progesterone (P4) and cortisol levels across three time periods: Baseline; Pre, (the week preceding the drop in P4); and Post, (the period after the P4 drop). Levels of P4 were significantly lower, and levels of cortisol were significantly higher in the days just prior to parturition. I found considerable intra- and inter-individual variation in both endocrine profiles, which can make endocrine assessments difficult to interpret in real time. In Chapter 3, I investigated whether behaviors in the preparturition period could be predictive of impending parturition in the Asian elephant. ANOVA results indicated a significant difference in the amount of time that elephants spent walking backwards across three time periods (F(2) = 3.723, p = 0.033), with the behavior increasing as parturition approached. These results were supported by a non-parametric Kruskal- Wallis. Using a generalized linear mixed model (GLMM), I found that as P4 levels decrease, walking backwards behavior significantly increases. In Chapter 4, I evaluated investigative trunk behaviors, or “trunk checks”, directed towards the temporal gland near the ear, mammary glands, vulva and anus of the pregnant dam. Investigative behaviors included both self-directed behaviors and those sent from herd mates towards the pregnant dam. Self-directed behaviors are most likely associated with physical changes in the pregnant dam, such as using the trunk to pull on swollen teats. Other-directed behaviors may stem from chemo-sensory signaling or other types of communication between herd mates, such as detecting changes in progesterone or cortisol. I ran GLMM and found that four trunk-check behaviors varied significantly with P4 and/or cortisol profiles. These were: self-checks of mammary glands increased with decreasing P4 levels; herd-mate-checks of mammary glands increased with decreasing P4 levels; self-checks of vulva increased with decreasing P4 levels and increasing cortisol levels; herd-mate-checks of anus increased with increasing cortisol levels. In Chapter 5, I evaluated activity budget behaviors in the pregnant elephants. Generalized comparisons were made to published activity budgets of typical captive Asian elephants. I report that activity budgets are within the range of normal activity, though I note a high level of inter-individual variation. In addition, I compared two sampling techniques, including one-zero and instantaneous sampling, that were used for activity budget data collection. I discuss the different results obtained by each sampling technique. These results are a very promising indication that behaviors, including walking backwards and multiple trunk-check behaviors, are changing over time or with parturition-related hormone profiles. We recommend that keepers, veterinary staff, and other observers that are familiar with the regular behavioral repertoire of a pregnant female should pay close attention to these highlighted behaviors. Keeping track of these behaviors, especially in conjunction with P4 and cortisol tracking, can help staff refine existing windows of expected parturition. Title: Predicting Parturition in a Long-Gestating Species: Behavioral and Hormonal Indicators in the Asian Elephant (Elephas maximus) Author: Velonis, Heather Kelly Year: 2017 Captive populations of Asian elephants (Elephas maximus) in North America are not self-sustaining, and increasing reproductive success within captive populations is a high priority. The ability to accurately predict parturition can have a direct impact on elephant welfare. Elephants in captivity often require significant preparation and management throughout the birthing process, and complications during labor and delivery can necessitate immediate intervention, including stillbirth, protracted labor, maternal aggression towards a newborn calf, and dystocia. Being able to predict when parturition will commence can ensure appropriate staff is available and adequate monitoring is performed. Routine endocrine sampling can be used to predict parturition in Asian elephants, with a drop in progesterone (P4) to baseline levels signaling parturition in 2-5 days. However, we determined this method is not without limitations, and it is not used in all institutions that house elephants. As changes in hormones regulate and alter behaviors, we investigated behavioral indicators as an additional management tool for predicting parturition, a time of drastic hormone changes. We conducted a study of five pregnancies in Asian elephants at the Oregon Zoo, U.S.A, and Taronga Zoo, Australia, between 2008 and 2012. In Chapter 2, I evaluated progesterone (P4) and cortisol levels across three time periods: Baseline; Pre, (the week preceding the drop in P4); and Post, (the period after the P4 drop). Levels of P4 were significantly lower, and levels of cortisol were significantly higher in the days just prior to parturition. I found considerable intra- and inter-individual variation in both endocrine profiles, which can make endocrine assessments difficult to interpret in real time. In Chapter 3, I investigated whether behaviors in the preparturition period could be predictive of impending parturition in the Asian elephant. ANOVA results indicated a significant difference in the amount of time that elephants spent walking backwards across three time periods (F(2) = 3.723, p = 0.033), with the behavior increasing as parturition approached. These results were supported by a non-parametric Kruskal- Wallis. Using a generalized linear mixed model (GLMM), I found that as P4 levels decrease, walking backwards behavior significantly increases. In Chapter 4, I evaluated investigative trunk behaviors, or “trunk checks”, directed towards the temporal gland near the ear, mammary glands, vulva and anus of the pregnant dam. Investigative behaviors included both self-directed behaviors and those sent from herd mates towards the pregnant dam. Self-directed behaviors are most likely associated with physical changes in the pregnant dam, such as using the trunk to pull on swollen teats. Other-directed behaviors may stem from chemo-sensory signaling or other types of communication between herd mates, such as detecting changes in progesterone or cortisol. I ran GLMM and found that four trunk-check behaviors varied significantly with P4 and/or cortisol profiles. These were: self-checks of mammary glands increased with decreasing P4 levels; herd-mate-checks of mammary glands increased with decreasing P4 levels; self-checks of vulva increased with decreasing P4 levels and increasing cortisol levels; herd-mate-checks of anus increased with increasing cortisol levels. In Chapter 5, I evaluated activity budget behaviors in the pregnant elephants. Generalized comparisons were made to published activity budgets of typical captive Asian elephants. I report that activity budgets are within the range of normal activity, though I note a high level of inter-individual variation. In addition, I compared two sampling techniques, including one-zero and instantaneous sampling, that were used for activity budget data collection. I discuss the different results obtained by each sampling technique. These results are a very promising indication that behaviors, including walking backwards and multiple trunk-check behaviors, are changing over time or with parturition-related hormone profiles. We recommend that keepers, veterinary staff, and other observers that are familiar with the regular behavioral repertoire of a pregnant female should pay close attention to these highlighted behaviors. Keeping track of these behaviors, especially in conjunction with P4 and cortisol tracking, can help staff refine existing windows of expected parturition. Close

• 48. [Article] Examination of Human Impacts on the Biodiversity and Ecology of Lichen and Moss Communities

  Globally, more than half of the world's population is living in urban areas and it is well accepted that human activities (e.g. climate warming, pollution, landscape homogenization) pose a multitude of ...

  Citation × Citation Citation Abstract Copy Title: Examination of Human Impacts on the Biodiversity and Ecology of Lichen and Moss Communities Author: Prather, Hannah Marie Year: 2017 Globally, more than half of the world's population is living in urban areas and it is well accepted that human activities (e.g. climate warming, pollution, landscape homogenization) pose a multitude of threats to ecosystems. Largely, human-related impacts on biodiversity will hold consequences for larger ecological processes and research looking into human impacts on sensitive epiphytic lichen and moss communities is an emerging area of research. While seemingly small, lichen and moss communities exist on nearly every terrestrial ecosystem on Earth and contribute to whole-system processes (e.g. hydrology, mineral cycling, food web energetics) worldwide. To further examine human impacts on epiphytic communities, I conducted three studies examining urbanization and climate warming effects on epiphytic lichen and moss biodiversity and ecology. In the first study I revisited a historic urban lichen community study to assess how urban lichen communities have responded to regional air quality changes occurring over the last nearly two decades. I further investigated, for the first time, the biodiversity of urban tree canopy-dwelling lichen communities in a native coniferous tree species, Pseudotsuga menziesii. I found that urban parks and forested areas harbor a species rich community of lichens epiphytes. Further, I found evidence for the distinct homogenization of urban epiphytic lichen communities, suggesting that expanding beyond simplistic measures of biodiversity to consider community composition and functional biodiversity may be necessary when assessing the ecology and potential ecosystem services of epiphyte communites within urbanizing landscapes. Next, I present the first tall tree canopy study across a regional gradient of urbanization near Portland, Oregon, USA. I found that tall tree canopy epiphyte communities change dramatically along gradients of increasing urbanization, most notably by the transitioning of species functional groups from sensitive, oligotrophic species to a dominance of urban-tolerant, eutrophic species. The implications these dramatic shifts in species composition have on essential PNW ecosystem processes, like N-fixation and canopy microclimate regulation, is still not well understood and is difficult to formally evaluate. However, I find strong evidence that native conifer trees in urban areas may provide a diversity of essential ecosystem services, including providing stratified habitat for epiphyte communities and their associated micro arthropod communities and the scavenging of atmospherically deposited nutrients. Future work is needed to understand how losses in canopy N fixation and species with large biomass (both lichens and bryophytes) will affect nutrient and hydrologic cycling in the PNW region, which continue to undergo rapid growth and urbanization. The final chapter investigates the impacts of passive warming by Open Top Chambers (OTCs) in moss-dominated ecosystems located on the Western Antarctic Peninsula, an area of increasing climate warming. I compared species-specific temperature effects, moss canopy morphology, sexual reproductive effort and invertebrate communities between OTC and control moss communities for two moss species, Polytrichastrum alpinum and Sanionia uncinata, that make up over 65% of the terrestrial vegetative cover in the area. I found distinct reproductive shifts in P. alpinum under passive warming compared to controls. Moss communities under warming also had substantially larger total invertebrate communities than those in control moss communities, and invertebrate communities were significantly affected by moss species and moss reproductive effort. Further, substantial species-specific thermal differences among contiguous patches of these dominant moss species were revealed. These results suggest that continued warming will differentially impact the reproductive output of Antarctic moss species and is likely to dramatically alter terrestrial ecosystems dynamics from the bottom up. This combined work provides a diverse contribution to the field of epiphyte ecology and biology by providing new insights on how human impacts will affect epiphyte lichen and moss communities across diverse ecosystems, in light of a rapidly changing planet. Title: Examination of Human Impacts on the Biodiversity and Ecology of Lichen and Moss Communities Author: Prather, Hannah Marie Year: 2017 Globally, more than half of the world's population is living in urban areas and it is well accepted that human activities (e.g. climate warming, pollution, landscape homogenization) pose a multitude of threats to ecosystems. Largely, human-related impacts on biodiversity will hold consequences for larger ecological processes and research looking into human impacts on sensitive epiphytic lichen and moss communities is an emerging area of research. While seemingly small, lichen and moss communities exist on nearly every terrestrial ecosystem on Earth and contribute to whole-system processes (e.g. hydrology, mineral cycling, food web energetics) worldwide. To further examine human impacts on epiphytic communities, I conducted three studies examining urbanization and climate warming effects on epiphytic lichen and moss biodiversity and ecology. In the first study I revisited a historic urban lichen community study to assess how urban lichen communities have responded to regional air quality changes occurring over the last nearly two decades. I further investigated, for the first time, the biodiversity of urban tree canopy-dwelling lichen communities in a native coniferous tree species, Pseudotsuga menziesii. I found that urban parks and forested areas harbor a species rich community of lichens epiphytes. Further, I found evidence for the distinct homogenization of urban epiphytic lichen communities, suggesting that expanding beyond simplistic measures of biodiversity to consider community composition and functional biodiversity may be necessary when assessing the ecology and potential ecosystem services of epiphyte communites within urbanizing landscapes. Next, I present the first tall tree canopy study across a regional gradient of urbanization near Portland, Oregon, USA. I found that tall tree canopy epiphyte communities change dramatically along gradients of increasing urbanization, most notably by the transitioning of species functional groups from sensitive, oligotrophic species to a dominance of urban-tolerant, eutrophic species. The implications these dramatic shifts in species composition have on essential PNW ecosystem processes, like N-fixation and canopy microclimate regulation, is still not well understood and is difficult to formally evaluate. However, I find strong evidence that native conifer trees in urban areas may provide a diversity of essential ecosystem services, including providing stratified habitat for epiphyte communities and their associated micro arthropod communities and the scavenging of atmospherically deposited nutrients. Future work is needed to understand how losses in canopy N fixation and species with large biomass (both lichens and bryophytes) will affect nutrient and hydrologic cycling in the PNW region, which continue to undergo rapid growth and urbanization. The final chapter investigates the impacts of passive warming by Open Top Chambers (OTCs) in moss-dominated ecosystems located on the Western Antarctic Peninsula, an area of increasing climate warming. I compared species-specific temperature effects, moss canopy morphology, sexual reproductive effort and invertebrate communities between OTC and control moss communities for two moss species, Polytrichastrum alpinum and Sanionia uncinata, that make up over 65% of the terrestrial vegetative cover in the area. I found distinct reproductive shifts in P. alpinum under passive warming compared to controls. Moss communities under warming also had substantially larger total invertebrate communities than those in control moss communities, and invertebrate communities were significantly affected by moss species and moss reproductive effort. Further, substantial species-specific thermal differences among contiguous patches of these dominant moss species were revealed. These results suggest that continued warming will differentially impact the reproductive output of Antarctic moss species and is likely to dramatically alter terrestrial ecosystems dynamics from the bottom up. This combined work provides a diverse contribution to the field of epiphyte ecology and biology by providing new insights on how human impacts will affect epiphyte lichen and moss communities across diverse ecosystems, in light of a rapidly changing planet. Close

• 49. [Article] THE ROLE OF CHROMODOMAIN PROTEIN 1 (CDP-1) AT NEUROSPORA CRASSA HETEROCHROMATIN

  36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2...

  Citation × Citation Citation Abstract Copy Title: THE ROLE OF CHROMODOMAIN PROTEIN 1 (CDP-1) AT NEUROSPORA CRASSA HETEROCHROMATIN Author: Kim, Carissa Year: 2016 36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2016. Title: THE ROLE OF CHROMODOMAIN PROTEIN 1 (CDP-1) AT NEUROSPORA CRASSA HETEROCHROMATIN Author: Kim, Carissa Year: 2016 36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2016. Close