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• 11. [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

• 12. [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

• 13. [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

• 14. [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

• 15. [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

• 16. [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

• 17. [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

• 18. [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

• 19. [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

• 20. [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