Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation.

Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation.

Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation. / Science, Faculty of / Zoology, Department of / Graduate

The soil-nematode Caenorhabditis elegans survives oxygen deprivation (anoxia < 0.001 kPa of O2, 0% O2) by entering into a state of suspended animation during which cell cycle progression at interphase, prophase and metaphase stage of mitosis is arrested. I conducted cell biological characterization of embryos exposed to various anoxia exposure times, to demonstrate the requirement and functional role of spindle checkpoint gene san-1 during brief anoxia exposure. I conducted a synthetic lethal screen, which has identified genetic interactions between san-1, other spindle checkpoint genes, and the kinetochore gene hcp-1. Furthermore, I investigated the genetic and cellular mechanisms involved in anoxia-induced prophase arrest, a hallmark of which includes chromosomes docked at the nuclear membrane. First, I conducted in vivo analysis of embryos carried inside the uterus of an adult and exposed to anoxic conditions. These studies demonstrated that anoxia exposure prevents nuclear envelope breakdown (NEBD) in prophase blastomeres. Second, I exposed C. elegans embryos to other conditions of mitotic stress such as microtubule depolymerizing agent nocodazole and mitochondrial inhibitor sodium azide. Results demonstrate that NEBD and chromosome docking are independent of microtubule function. Additionally, unlike anoxia, exposure to sodium azide causes chromosome docking in prophase blastomeres but severely affects embryonic viability. Finally, to identify the genetic mechanism(s) of anoxia-induced prophase arrest, I conducted extensive RNA interference (RNAi) screen of a subset of kinetochore and inner nuclear membrane genes. RNAi analysis has identified the novel role of 2 nucleoporins in anoxia-induced prophase arrest.

An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous to human genes that are differentially regulated in response to metabolic diseases, suggesting that there may be conserved gene expression responses between C. elegans supplemented with glucose and a diabetic and/or obese state observed in humans. These findings support the utility of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-induced sensitivity to oxygen deprivation) and identify potentially novel regulators of common complications seen in hyperglycemic and T2D patients (e.g., macrovascular complications).

Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. elegans. Furthermore, these studies have begun to uncouple mechanisms of longevity and stress resistance.

O estrógeno é importante para o desenvolvimento de redes neuronais. Assim, investigamos mecanismos celulares relacionados à neuroproteção, através da sinalização rápida mediada pelo GPER-1 em cultura mistas e enriquecida de neurônios submetidas ou não à privação de glicose/oxigênio (PGO). Mostramos que as células corticais em cultura expressam o receptor GPER-1 e esta marcação encontra-se dispersa tanto no citosol como no núcleo. Nossos resultados mostraram que a proteção, via sinalização estrogênica, foi dependente da composição celular. A ausência da sinalização via GPER-1 previamente à PGO aumentou a morte celular induzida pela PGO, sugerindo que o bloqueio desta sinalização via GPER-1 pode estar relacionado ao pior prognóstico de lesões isquêmicas, e a suplementação com G1 no meio de cultura durante a privação e reperfusão atenuaram estes efeitos. Além disso, nossos resultados apontam para a influência das células da glia como mediadores do papel neuroprotetor, via sinalização estrogênica não-nuclear, neste contexto de privação de glicose/oxigênio. / Estrogen is important to the development of neural networks. Thus, we investigated the cellular mechanisms related to neuroprotection through the rapid signaling mediated by GPER-1 in mixed culture and enriched neurons submitted or not to glucose/oxygen deprivation (OGD). We showed that cortical cell cultures express GPER-1 receptor and this are dispersed both in the cytosol and the nucleus. Our results showed that protection via estrogen signaling was dependent on the cellular composition. The lack of a signaling pathway GPER-1 before OGD increased cell death induced by OGD, suggesting that blocking of GPER-1 signaling pathway could be related to poor prognosis of ischemic lesions and G1 supplementation of culture media during deprivation and reperfusion attenuated these effects. In addition, our results point to the influence of glial cells as mediators of the neuroprotective role via non-nuclear estrogen signaling in this context of glucose/oxygen deprivation.

O estrógeno é importante para o desenvolvimento de redes neuronais. Assim, investigamos mecanismos celulares relacionados à neuroproteção, através da sinalização rápida mediada pelo GPER-1 em cultura mistas e enriquecida de neurônios submetidas ou não à privação de glicose/oxigênio (PGO). Mostramos que as células corticais em cultura expressam o receptor GPER-1 e esta marcação encontra-se dispersa tanto no citosol como no núcleo. Nossos resultados mostraram que a proteção, via sinalização estrogênica, foi dependente da composição celular. A ausência da sinalização via GPER-1 previamente à PGO aumentou a morte celular induzida pela PGO, sugerindo que o bloqueio desta sinalização via GPER-1 pode estar relacionado ao pior prognóstico de lesões isquêmicas, e a suplementação com G1 no meio de cultura durante a privação e reperfusão atenuaram estes efeitos. Além disso, nossos resultados apontam para a influência das células da glia como mediadores do papel neuroprotetor, via sinalização estrogênica não-nuclear, neste contexto de privação de glicose/oxigênio. / Estrogen is important to the development of neural networks. Thus, we investigated the cellular mechanisms related to neuroprotection through the rapid signaling mediated by GPER-1 in mixed culture and enriched neurons submitted or not to glucose/oxygen deprivation (OGD). We showed that cortical cell cultures express GPER-1 receptor and this are dispersed both in the cytosol and the nucleus. Our results showed that protection via estrogen signaling was dependent on the cellular composition. The lack of a signaling pathway GPER-1 before OGD increased cell death induced by OGD, suggesting that blocking of GPER-1 signaling pathway could be related to poor prognosis of ischemic lesions and G1 supplementation of culture media during deprivation and reperfusion attenuated these effects. In addition, our results point to the influence of glial cells as mediators of the neuroprotective role via non-nuclear estrogen signaling in this context of glucose/oxygen deprivation.