Ventilatory drive is mediated by respiratory central pattern generators that are located in the brainstem, which are continuously modulated by specialised peripheral and central chemoreceptors to adjust ventilatory patterns according to changes in arterial PO2. These specialised oxygen-sensing chemoreceptors are activated in response to acute reductions in arterial PO2 and ultimately trigger a respiratory response that acts to restore oxygen-levels. However, the molecular mechanism by which mammals are able to regulate their breathing pattern in such a manner during hypoxia remains controversial. Therefore, the studies performed in this thesis aimed to investigate the possibility that this process may be mediated by the liver kinase B 1 (LKB1)/ AMP-activated protein kinase (AMPK) signalling pathway, which is central to cellular adaptations to metabolic stress. This first involved the development of transgenic mice in which Lkb1 or AMPK were deleted. Global knockout of Lkb1 (Sakamoto, 2006) or AMPK activity (Viollet et al., 2009) are embryonic lethal. Thus, the Cre/loxP system was used to develop transgenic mice that had either Lkb1 or both isoforms of the AMPK catalytic α- subunit (α1 and α2) conditionally knocked out in catecholaminergic cells (including therein hypoxia-activated cells of the brainstem and carotid body) by driving Cre expression through a tyrosine-hydroxylase-specific promoter region. The consequent effects on the ventilatory response to hypoxia were then examined using unrestrained whole-body plethysmography. This demonstrated that, in contrast to the hyperventilation evoked in controls, increased ventilation was virtually abolished in the Lkb1 and AMPK α1 and α2 double knockouts during hypoxia. Both knockout mice also exhibited periods of hypoventilation with frequent apnoeas during hypoxia. Additionally, studies on single AMPK α1 and AMPK α2 knockouts identified that the ventilatory dysfunction in AMPK α1 and α2 double knockouts was primarily caused by AMPK α1 deletion. In contrast, the severe ventilatory abnormalities exhibited during hypoxia following the deletion of Lkb1 and AMPK in catecholaminergic cells were mostly reversed upon exposure of mice to hypoxia with hypercapnia. Also, the ventilatory response to hypercapnia alone was without any major effect as a result of Lkb1 deletion or the dual-deletion of AMPK α1 and α2 catalytic subunits in catecholaminergic cells. This thesis therefore demonstrates, for the first time, that the LKB1-AMPK signalling pathway is key to respiratory adaptations during hypoxia, by regulating catecholaminergic oxygen-sensing cells, thus protecting against hypoventilation and apnoeas. The LKB1-AMPK signaling pathway can thereby determine oxygen and energy supply at both a cellular and whole-body level.

Transcriptional control plays a major role in regulating hypoxic responses in plants. However, the transcriptional regulatory networks associated with hypoxia remain to be constructed. By transcriptomic analysis I show here that a novel systemic transcriptional reprogramming, which is mediated via the interplay of hormones, facilitates the survival of plants under flooding. A feasible strategy for identifying downstream targets of transcription factors (TFs) was developed. The downstream pathways of a hypoxia-responsive TF, WRKY22, were constructed. The results also show that AtERF73/HRE1 (Arabidopsis thaliana Ethylene Response Factor 73/Hypoxia Responsive ERF 1) modulate ethylene-dependent and -independent responses during hypoxia. Transcriptomic analysis of Arabidopsis in both root and shoot tissues during flooding of roots indicates the existence of a systemic communication through transcriptional reprogramming. By functional classification of affected genes, a comprehensive managing program of carbohydrate metabolism was observed. Through transcriptional profiling in ethylene and abscisic acid (ABA) signaling mutants, ein2-5 and abi4-1, an alteration of long-distance hypoxic regulation was uncovered in ein2-5 and abi4-1. Moreover, genes involved in ABA biosynthesis were also found to be differentially regulated between shoots and roots. Many members of the WRKY TF family were highly induced by hypoxia. One of the early-induced WRKYs, WRKY22, which has the highest induced level, was chosen for identifying its downstream targets. Anoxic tolerance was affected in WRKY22 overexpressing (WRKY22-OX) and knock-out (wrky22-ko) lines. Comparison of differential gene expression profiles between the wild-type and WRKY22-OX and between the wild-type and wrky22-ko lines by microarray analysis identified novel hypoxia-responsive genes as WRKY22 targets. Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) and ChIP followed by quantitative PCR (ChIP-qPCR) were utilized to analyze in vivo interactions. To study the role of ethylene during hypoxia, I characterized an AP2/ERF (APETALA2/ethylene response factor) AtERF73/HRE1 that is specifically induced during hypoxia. I showed that the expression of AtERF73/HRE1 can be induced by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. Its hypoxic induction was reduced but not completely abolished in ethylene-insensitive mutants and in the presence of inhibitors of ethylene biosynthesis and responses. Increased ethylene sensitivity and exaggerated triple responses were observed in HRE1-RNAi knock-down lines. By comparing expression differences between the wild-type and HRE1-RNAi lines, I found that hypoxic induction of glycolytic and fermentative genes was reduced by the HRE1-RNAi knock-down mutations, whereas induction of a number of peroxidase and cytochrome P450 genes was increased. Collectively, these results show that AtERF73/HRE1 is involved in modulating ethylene responses under both normoxia and hypoxia.

An essential event during early pregnancy is the invasion of trophoblasts into the maternal decidua, which is necessary for proper implantation and establishment of maternal-fetal interface and ultimately allows for proper nutrient exchange and immunological tolerance of the growing fetus. For this invasion to occur, cells originating from the trophectoderm undergo an epithelial to mesenchymal transition to become invasive extravillous trophoblasts and begin invading the uterine decidual tissue. Through the secretion of matrix metalloproteinases and through interactions with many cytokines and cell-adhesion molecules, this well-orchestrated process of trophoblast invasion results in extensive remodeling of the maternal spiral vasculature by the extravillous trophoblasts. Ultimately, the spiral arteries are transformed from high resistance, low flow vessels to low resistance, high flow vessels to allow for adequate perfusion of the placenta and developing fetus. Preeclampsia is a leading cause of maternal morbidity worldwide and is associated with the onset of hypertension and proteinuria, typically after 20 weeks of gestation. While the hypertension typically resolves following delivery of the fetus and placenta, both the mother and growing child are faced with long-term adverse health effects such as the development of cardiovascular disease and metabolic disorders. Preeclampsia is characterized by widespread maternal inflammation and endothelial dysfunction triggered by the secretion of soluble factors from the placenta into the maternal circulation. It is thought that the onset of these adverse systemic conditions is initiated by poor placental perfusion and pathologically hypoxic conditions in the placenta. In many cases of preeclampsia, there is evidence of shallow trophoblast invasion which results in incomplete spiral artery transformation, ultimately leading to poor placental perfusion. However, the exact mechanisms underlying the inadequate extravillous trophoblast invasion and remodeling are incompletely understood. LIN28 is an RNA binding protein that is highly expressed in embryonic stem cells, fetal tissues and many cancers, and was discovered as a regulator of the maturation of the Let-7 family of miRNAs. However, as an RNA binding protein, LIN28 has been shown to interact with thousands of mRNA transcripts, leading to both increased and decreased protein expression, and control of many cellular processes such as differentiation, proliferation, migration, invasion, and cellular metabolism. In vertebrates, LIN28 exists as two highly homologous paralogs, LIN28A and LIN28B, however LIN28B is slightly larger and contains a nuclear localization signal not found in LIN28A. While they both function to inhibit Let-7 maturation, there is evidence to suggest they also have independent functions. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to preeclampsia. We found that LIN28B mRNA expression is ~1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in human primary cytotrophoblasts, syncytiotrophoblasts, and decidual cells. We also found that LIN28B mRNA and protein levels are significantly reduced in human placentas from preeclamptic pregnancies compared to placentas from normal pregnancies, while LIN28A expression is unchanged. Upon investigation of human first trimester placenta sections, we found that LIN28B is more highly expressed in the invasive extravillous trophoblasts and syncytial sprouts compared to villous trophoblasts. To support this with in vitro evidence, we found that overexpression of LIN28B in human HTR8/SVneo cells resulted in increased proliferation, migration and invasion, while knockdown of LIN28B in JEG3 cells resulted in decreased proliferation. Furthermore, knockdown of LIN28B in JEG3 cells led to decreased expression of SYN-1, ELABELA, and the chromosome 19 miRNA cluster, with accompanying increases in the pro-inflammatory cytokine TNFα and ITGβ4, an integrin enriched on non-invasive trophoblasts. Moreover, culture of JEG3 and BEWO cells in hypoxia resulted in significantly decreased levels of LIN28B mRNA and protein expression, as well as syncytin-1 and ELABELA mRNA levels, while TNFα was increased. These results provide the first evidence that LIN28B is the predominant paralog expressed in human placenta and decreased LIN28B may play a crucial role in PE pathogenesis by reducing trophoblast invasion, syncytialization and by promoting inflammation.

Background: Perinatal ischemic brain injury leads to developmental disability (DD), which accounts for 30% of disabilities in children. Antepartum risk, or risk occurring prior to birth occurs in more than 90% of cases. This study investigated whether maternal ingestion of a natural health product (broccoli sprouts) would provide neuroprotection in an intrauterine model of HI. Methods: Intrauterine ischemia was induced by bilateral uterine artery ligation (BUAL) on E20 of gestation. Rats were fed broccoli sprouts (200 mg) from E15 until postnatal day 14 (PD14). Rat pups underwent neurobehavioural testing from birth to PD21 and were then sacrificed for neuropathologic assessment on PD21. Results: BUAL ligation resulted in growth restriction (IUGR) of the fetuses, which persisted throughout the study (p < 0.001). Reflex testing indicated IUGR pups were developmentally delayed compared to controls (p < 0.001). Open field testing on PD21 indicated hyperactivity in IUGR animals compared to controls (p < 0.001). Histological assessment showed a reduction in pyramidal cells in CA1 and CA3 of IUGR hippocampi and in myelin basic protein (MBP) immunohistochemistry signal. Broccoli sprout supplementation improved some reflex and behavioural measures, increased cell counts in CA1 and CA3 as well as MBP signal in growth restricted animals. Conclusions: Supplementation with broccoli sprouts during the last trimester of gestation and the first 2 weeks of life in the rat lessened the effects of chronic intra-uterine ischemia. These findings suggest a novel approach to the prevention of DD associated with perinatal HI.

Sildenafil increases oxygen delivery and maximal exercise capacity at very high altitudes (greater than or equal to 4300 m) and has been shown to improve short-duration exercise performance in some individuals at simulated high altitude (3900 m). It is unknown whether sildenafil improves maximal exercise capacity and longer duration exercise performance at moderate and high altitudes where competitions are more common. Additionally, the effects of sildenafil on women exercising at altitude have not been examined. The purpose of this study was to determine the effects of sildenafil on cardiovascular hemodynamics, arterial oxygen saturation (SaO2), peak exercise capacity (Wpeak), and 15-km time trial performance, in endurance-trained men and women at simulated moderate (MA; 2100 m, 16.2 % FIO2) and high (HA; 3900 m, 12.8% FIO2) altitudes. Eleven male and 10 female subjects completed two HA Wpeak trials following the ingestion of placebo or 50 mg sildenafil in randomized, counterbalanced, and double blind fashion. Subjects then completed four exercise trials (30 min at 55% of Wpeak + 15-km time trial) at MA and HA following the ingestion of placebo or 50 mg sildenafil in randomized, counterbalanced, and double blind fashion. Sildenafil had little influence on cardiovascular hemodynamics for either gender at MA or HA, but did result in higher SaO2 values compared to placebo during steady state and time trial exercise in men at HA only. Sildenafil did not affect Wpeak or 15-km time trial performance in either gender at MA or HA. We conclude that sildenafil is unlikely to exert beneficial effects at altitudes < 4000 m for a majority of the population.

Hypoxia in the marine environment is a growing environmental concern, and can have profound impacts on organisms. This dissertation seeks to understand the physiologically induced changes in gene expression, the relationship between gene expression and metabolism, and how these parameters vary among populations, in response to hypoxic stress. By comparing evolved intraspecific variation in gene expression and physiological parameters among populations from multiple regions in the Gulf of Mexico we seek to determine the physiologically induced changes that are essential to hypoxic survival. First, whole body metabolism, measured as oxygen uptake, was profiled across seven decreasing oxygen concentrations. Metabolism and the critical oxygen tension (PO2crit) were compared between populations from across the Gulf of Mexico. This study demonstrated a significant interaction of body mass with the hypoxic response. Additionally, populations only differed in their metabolism at the lowest oxygen concentration, 1.8 kPa. PO2crit did not differ between populations, but was body mass dependant. Next, the effects of hypoxia on gene expression were examined. These studies examined the effects of hypoxia on gene expression over time and at different hypoxic doses, utilizing a 384 gene microarray. In the first studies individuals were subjected to 0, 4, 8, 12, 24, 48, or 96 hours of hypoxia. Different genes had different times for peak gene expression, with most changes occurring after 96 hours of exposure. However, only 14 genes had significant changes in gene expression. To determine the effect of differing hypoxic dose, individuals were exposed to normoxia, 7.8 kPa O2 (moderate hypoxia), or 1.8 kPa (severe hypoxia) for 4 or 48 hours. Sixty-nine genes had significant changes in gene expression for either dose or time. To elucidate the relationship between effect of time and dose, genes were examined for dose response within each time. The maximum number of changes occurred at 1.8 kPa after 48 hours of exposure. Interestingly different sets of genes had changes in gene expression at either 7.8 or 1.8 kPa. Finally, to ascertain the difference among populations, for thousands of genes, individuals from six populations of Fundulus grandis were exposed to hypoxia (1.8 kPa) for 4 or 96 hours. Hypoxia had a significant effect on the expression of 609 genes, while population affected the expression of 355 genes. Genes with significant differences in expression among populations reflect geographic separation. For the 59 genes with significant differences in expression for both hypoxia response and population, shared hypoxic histories appears to be more important than simply the neutral patterns expected with geographic distance. The majority of significant changes for the 609 hypoxia responsive genes take place after 96 hours of hypoxia exposure. This research demonstrates that F. grandis cope with hypoxia through changes in metabolism and gene expression. Overall, the response to hypoxia is dependent on an individual's size (body mass), the ambient oxygen concentration, and the duration of hypoxia exposure. Additionally, there appear to be some differences between populations with differing exposure history to hypoxia in the Gulf of Mexico.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Timothy E. Targett, College of Marine and Earth Studies. Includes bibliographical references.

Bottom water hypoxia and its influence on the environment have been topics of increasing concern for many coastal regions. This research addresses both spatial and temporal variability in sediment biogeochemistry at the southeastern region of Corpus Christi Bay, TX, where seasonal (summer) hypoxia occurs. Traditional techniques for determination of a variety of dissolved and solid components, benthic oxygen demand, and sulfate reduction rates were augmented by measurements using solid state microelectrodes to simultaneously determine concentrations of dissolved O2, Mn2+, Fe2+, and [sigma]H2S in multiple small - interval (1 mm) depth profiles of sediment microcosms. Oxygen concentrations in the overlying water were manipulated in the sediment microcosms and electrode depth profile measurements were made over ~ 500 hours of experimentation. Laboratory and field microelectrode results were in good agreement for both norm - oxic and anoxic time periods. Results indicated that iron (Fe2+) and sulfide ([sigma]H2S) were the redox reactive species in these sediments. During hypoxic conditions an upward migration of dissolved Fe2+and [sigma]H2S through the sediment column and, at times, into the overlying water was observed as the dissolved oxygen concentrations decreased. A corresponding decline in the vertical extent of these redox species occurred when the overlying water was re-oxidized. When both dissolved iron and sulfide coexisted, FeS minerals were formed in the sediment, preventing sulfide diffusion into the overlying water. However, after a long duration of hypoxia (> 200 hours) this buffering capacity was exceeded and both iron and sulfide penetrated into the overlying waters. Results indicated that iron may have a greater influence on hypoxia than sulfide because its concentration in the overlying waters during induced hypoxia was an order of magnitude greater than those of sulfide. Moreover, in the southeastern region of the Bay, where mixing was minimal and the water column was shallow, the sediments alone may have caused the onset of the hypoxic event in a relatively short time period (< 5.5 days). These results demonstrated that in shallow marine environments where seasonal hypoxia occurs, such as Corpus Christi Bay, the associated major changes that take place in the sediment biogeochemistry must be included in benthic - pelagic models for overlying water hypoxia.

Hypoxia (dissolved oxygen concentrations less than 1.4 ml/l) is a recurrent seasonal phenomenon on the Louisiana Shelf, caused by the combined effects of nutrient loading by the Mississippi and Atchafalaya River System (MARS), and density stratification. In 2004, three shelf wide cruises (in April, June and August) were conducted on the Louisiana Shelf to understand the mechanisms controlling hypoxia on the shelf, and examine the relationship between dissolved oxygen and nutrient concentrations during the hypoxic periods. The shelf was divided into three geographically separate zones: A (off the mouth of the Mississippi River), B (off the Terrebonne Bay) and C (off the mouth of the Atchafalaya). Each zone was different in terms of the physical and biochemical processes occurring there. In April, no hypoxia was observed on the shelf because of water column mixing by winds, even though high discharge occurred from the MARS. Nutrients were abundant in the surface waters but present only in little amounts at the bottom. In June, the water column was highly stratified. Because of the presence of upwelling favorable winds no vertical mixing occurred and caused extensive hypoxia on the shelf. Dissolved oxygen concentrations were negatively correlated with nutrients at the bottom of the water column. Nutrients were considerably higher at the bottom than at the surface (except for zone A, where high nutrients were seen even at the bottom), indicating remineralization below the pycnocline. Resuspension of organic material and remineralized nitrogen were sustaining hypoxia far from the river sources. In August, hypoxia was patchy on the Louisiana Shelf. Correlations between dissolved oxygen and nutrient concentration varied seasonally with highest correlations occurring during hypoxic conditions in June and August. The spatial distribution of nutrients and other oceanographic parameters, such as light transmission, fluorescence, and dissolved oxygen concentrations, indicate seasonal variability of biochemical processes that are related to physical processes that affect stratification.

No description available.