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Introduction – Severe pulmonary bacterial infections are frequently complicated by systemic hypoxaemia and, in the context of acute respiratory distress syndrome (ARDS), inappropriately prolonged neutrophilic inflammation. This combination of acute hypoxaemia and persistent inflammatory response carries significant morbidity and mortality. However, patients with chronic lung disease function in the community with chronic systemic hypoxaemia and bacterial colonisation with much lower acute mortality. The HIF/PHD pathway tightly regulates neutrophilic responses to hypoxia and bacteria. Here, using acute bacterial pneumonia models, I have dissected the differences in innate immune responses to infection in acute hypoxia and following exposure to hypoxia prior to infection (‘preconditioning’). Methods – C57BL/6 mice were housed in room air or ‘preconditioned’ by exposure to 10% ambient hypoxia for seven days. They were then instilled with intratracheal Streptococcus pneumoniae (1x104 or 1x107 cfu to assess macrophage and neutrophil function respectively) under recovery anaesthesia and housed in normoxia (21% O2) or hypoxia (10% O2). At pre-determined time-points, the animals were assessed clinically for sickness and rectal temperature. Blood, bronchoalveolar lavage and tissues were taken for analysis. Transcriptome analysis by RNA-sequencing and functional glycolysis by Seahorse was performed on blood leucocytes. Results – Concurrent exposure to hypoxia and infection resulted in neutrophil-mediated morbidity and mortality. Acute hypoxia caused rapid utilisation of glucose, glycogen and fat stores resulting in systemic hypoglycaemia and death. Preconditioning with exposure to hypoxia prior to infection completely protected the host against hypoxia-induced morbidity and mortality by suppressing leucocyte glycolysis, through suppression of HIF1α, and resultant rescue from the negative energy state and cardiovascular compromise. Conclusion – Hypoxia preconditions the innate immune response by suppression of HIF1α and glycolysis in leucocytes, thereby protecting against acute hypoxia-induced mortality outcomes in acute bacterial pulmonary infection.

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.

Deflocculation is a common upset event in biological wastewater treatment plants and causes significant problems in biosolids discharge and environmental management. However, fundamental understanding of deflocculation is limited. The overall objective of this work was to explore the fundamentals for deflocculation under transients of short-term low dissolved oxygen (DO). The investigation was carried out in a sequence of batch and continuous experiments on activated sludge, followed by batch experiments on E. coli suspensions. Both batch and continuous experiments on activated sludge demonstrated deflocculation of bioflocs under the transients of low DO (< 0.5 mg/L). Under the short-term low DO (in hours), turbidity increased by 20 times in the batch system and by 1-2 times in the continuous system, concentrations of suspended solids increased by 1-2 times, number of small particles (< 12.5 mm) increased by 2 times, more soluble EPS (proteins and humic substances) were released into supernatant or treated effluents, the removal efficiency of organic compounds was reduced by 50-70%. A 40% of increase in bulk K+ but a 30% of decrease in bulk Ca2+ under the DO limitation were observed in the batch experiments. There were significant increases in bulk K+ and decreases in bulk Ca2+ in the continuous experiments. Reversible changes were observed within 24 hours once the DO stress was removed. Floc strength of the remaining bioflocs after deflocculation increased. Deflocculation under the short-term low DO was consistent with an erosion process. The addition of selected chemicals (i.e., Ca2+, tetraethylammonium chloride, glibenclamide, and valinomycin) did not prevent deflocculation under the short-term low DO. It is proposed that a DO stress causes an efflux of cellular K+ but an influx of extracellular Ca2+, resulting in a decreasing ratio of Ca2+/K+ in extracellular solution and thereby causing deflocculation. The E. coli tests supported that increasing bulk K+ under the DO limit was due to the release of cellular K+ and was a stress response to the DO limitation.

Deflocculation is a common upset event in biological wastewater treatment plants and causes significant problems in biosolids discharge and environmental management. However, fundamental understanding of deflocculation is limited. The overall objective of this work was to explore the fundamentals for deflocculation under transients of short-term low dissolved oxygen (DO). The investigation was carried out in a sequence of batch and continuous experiments on activated sludge, followed by batch experiments on E. coli suspensions. Both batch and continuous experiments on activated sludge demonstrated deflocculation of bioflocs under the transients of low DO (< 0.5 mg/L). Under the short-term low DO (in hours), turbidity increased by 20 times in the batch system and by 1-2 times in the continuous system, concentrations of suspended solids increased by 1-2 times, number of small particles (< 12.5 mm) increased by 2 times, more soluble EPS (proteins and humic substances) were released into supernatant or treated effluents, the removal efficiency of organic compounds was reduced by 50-70%. A 40% of increase in bulk K+ but a 30% of decrease in bulk Ca2+ under the DO limitation were observed in the batch experiments. There were significant increases in bulk K+ and decreases in bulk Ca2+ in the continuous experiments. Reversible changes were observed within 24 hours once the DO stress was removed. Floc strength of the remaining bioflocs after deflocculation increased. Deflocculation under the short-term low DO was consistent with an erosion process. The addition of selected chemicals (i.e., Ca2+, tetraethylammonium chloride, glibenclamide, and valinomycin) did not prevent deflocculation under the short-term low DO. It is proposed that a DO stress causes an efflux of cellular K+ but an influx of extracellular Ca2+, resulting in a decreasing ratio of Ca2+/K+ in extracellular solution and thereby causing deflocculation. The E. coli tests supported that increasing bulk K+ under the DO limit was due to the release of cellular K+ and was a stress response to the DO limitation.

Paratetramitus jugosus, a vahlkampfiid amoebomastigote, was isolated into monoprotist/monobacterial (Bacillus sp.), cultures from laminated microbial mats (Laguna Figueroa, Baja California Norte, Mexico) and muds (Eel Pond, Woods Hole, Massachusetts). Chromidia, roughly spherical (2-4 µm in diameter) were released from both walled spherical cysts (10-12 µm) and phagocytotic amoebic forms. Desiccation-resistant walled chromidia, at first spherical, resorb their walls and develop into small pleiomorphic phagocytotic amoeba. Small amoebae feed and mature into typical monopodial vahlkampfiid adults confirming previous work (Dobell, 1913, and especially the analysis of a larger encysting vahlkampfiid amoeba associated with Long Island oyster disease studied at Woods Hole by Mary Jane Hogue, 1914). I show here that P. jugosus reproduces and develops through its life history stages of chromidia, mature monopodial amoebae, and cysts as rapidly and abundantly under low oxygen levels as at ambient atmospheric oxygen concentrations. Anoxia was achieved in the laboratory by incubation of entirely desiccated inocula from old mat or mud samples in Brewer jars with or without gas packs to control atmospheric conditions. Three sets of experiments yielded the same results: vigorous growth on bacillus food occurred on manganese acetate media by two weeks on the surface of agar plates under ambient oxic or hypoxic to anoxic conditions. Preliminary investigations of similar amoeba from geographically distinct field sites in Europe, North America, and the Caribbean were made. From them, I suggest it is likely these coastal amoebomastigotes that propagate by small desiccation resistant, oxygen-independent, manganese tolerant chromidia are genuinely cosmopolitan in its distribution.

This thesis investigates the effects of low oxygen culture conditions and fibroblast growth factor-2 (FGF2) on adult human dermal fibroblasts. It was previously shown that low oxygen and FGF2 culture conditions lead to an extension of proliferative lifespan, low-level activation of stem cell genes, and global transcriptional changes in adult human dermal fibroblasts. Additionally, an increased in vivo tissue regenerative response can be observed when human muscle-derived fibroblasts grown with FGF2 and low oxygen are implanted into mouse muscle injury, leading to a decrease in collagen deposition and scar formation and increase of functional skeletal muscle regeneration, including formation of Pax7+ muscle stem cells. These findings led to an analysis of key cellular oxygen sensors, hypoxia inducible factors (HIFs) and their role in this regenerative response. Directly linking these factors with the regenerative response, I have shown, with knockdown experiments, that HIF-2α is required for the increased proliferative capability and decreased senescence of human dermal fibroblasts (hDFs) induced by hypoxia. I have also determined that low oxygen causes an early and transient increase of HIF-1α and late and sustained increase of HIF-2α protein accompanied by increased nuclear translocation. Using overexpression and knockdown approaches via lent-virus, I determined that HIF-2α appears to modulate FGF2 signaling through the FGF receptors. First, under low oxygen conditions, exogenous FGF2 led to downregulation of endogenous FGF2, which can be mimicked by overexpression of HIF-2α. In ambient oxygen we didn't see this effect. Second, HIF-2α overexpression appears to lead to increases in FGFR1 phosphorylation and consequently increased ERK1/2 phosphorylation, and increases in the expression of heparan sulfate modifying enzymes (NDST1, NDST2, and EXTL2). Lastly, sustained supplementation with FGF2 in low oxygen inhibits receptor-mediated FGF2 signaling. To understand these effects at the transcriptional level, using microarray technology, we identified oxygen-mediated FGF2 effects on genes involved in cell survival and proliferation. Through bioinformatics analyses, I determined that genes involved in wound healing (extracellular matrix genes, adhesion molecules, cytokines) are upregulated in FGF2 treated fibroblasts grown under low oxygen. By utilizing a gain-of-function approach, we were able to assess the effects of altered HIF-2α activity on the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 in adult hDFs. The results indicate that overexpression of the HIF-2α transcription factor increases Oct4 mRNA, but not Oct4 protein, levels, and had no effect on Nanog and Lin28 proteins. HIF-2α overexpression also mediated FGF2 induction of Sox2 and Rex1 proteins of higher molecular weight. This thesis expands our knowledge about effects of low oxygen and FGF2 on adult human dermal fibroblasts and explains in part, how FGF2 under low oxygen conditions may lead to increased proliferation, extended life span, regenerative competency and increased developmental plasticity of adult hDFs.

Moderate or Intense Low-oxygen Dilution (MILD) combustion is one of combustion technologies which can improve efficiency and reduce emissions simultaneously. This combustion type is characterised by the highly preheated reactant temperature and the relatively small temperature rise during combustion due to the intense dilution of the reactant mixture. These unique combustion conditions give MILD combustion very attractive features such as high combustion efficiency, reduction of pollutant emissions, attenuation of combustion instabilities and flexibility of the flow field. However, our understanding of MILD combustion is not enough to employ the MILD combustion technology further for modern combustion devices. In this thesis, Direct Numerical Simulation (DNS) has been carried out for turbulent MILD combustion under four MILD and classical premixed conditions. A two-phase strategy is employed in the DNS to include the effect of imperfect mixing between fresh and exhaust gases before intense chemical reactions start. In the simulated instantaneous MILD reaction rate fields, both thin and distributed reaction zones are observed. Thin reaction zones having flamelet like characteristics propagate until colliding with other thin reaction zones to produce distributed reaction zones. Also, the effect of such interacting reaction zones on scalar gradient has to be taken into account in flamelet approaches. Morphological features of MILD reaction zones are investigated by employing Minkowski functionals and shapefinders. Although a few local reaction zones are classified as thin shape, the majority of local reaction zones have pancake or tube-like shapes. The representative scales computed by the shapefinders also show a typical volume where intense reactions appear. Given high temperature and existence of radicals in the diluted reactants, both reaction dominated and flame-propagation dominated regions are locally observed. These two phenomena are closely entangled under a high dilution condition. The favourable conditions for these phenomena are investigated by focusing on scalar fluxes and reaction rate. A conditional Probability Density Function (PDF) is proposed to investigate flamelet/non-flamelet characteristics of MILD combustion. The PDF can be obtained by both numerically and experimentally. The PDF shows that MILD combustion still has the direct relationship between reaction rate and scalar gradient, although the tendency is statistically weak due to the distributed nature of MILD reaction zones. Finally, based on the physical aspects of MILD combustion explained in this work, a representative model reactor for MILD combustion is developed. The model reactor is also used in conjunction with the presumed PDF for a mean and filtered reaction rate closure. The results show a good agreement between the modelled reaction rate and the DNS results.

Traditionally, hypoxia has been defined as the situation where DO levels have fallen below 2.0 mg O2 L-1, but increasing evidence suggests that this low level of DO is inadequate to describe the onset of hypoxia impacts for many organisms. Consequently, there is a need for a greater understanding of how ‘moderate’ alterations in DO levels will affect ecosystem processes and functionality, specifically through behavioural and physiological alterations at the organism and community level. This thesis reports on mesocosm experiments which were conducted to examine the effects of moderate ( > 3.0 mg O2 L-1) hypoxia on firstly, a key ecosystem engineer, the brittlestar Amphiura filiformis, and secondly, on the Station L4 infaunal macrobenthic community. Station L4 is a longstanding marine biodiversity and MSFD reference site and forms part of the Western Channel Observatory. At the organism level, short-term (14 d) exposure to moderate hypoxia significantly reduced oxygen uptake rates, oocyte diameter and oocyte development in A. filiformis. However, these physiological affects occurred irrespective of brittlestar population density. Additionally, moderate hypoxia reduced brittlestar activity, in terms of bioturbation behaviour, consequentially having an effect on ammonium and silicate fluxes. These observations were only detected when brittlestar population density was high. It was concluded that denser populations of A. filiformis may therefore exhibit the greatest changes in behaviour and shifts in ecosystem function as competition for resources and oxygen heightens. The benthic community at Station L4, displayed considerable tolerance to medium-term (6 wk.) exposure to moderate hypoxia, in terms of structure, diversity and bioturbatory behaviour, but these results may be different if exposure was longer or more severe. Alterations in nutrient fluxes were detected, but there was little evidence to suggest these changes were due to macrofaunal behavioural alterations. Additionally, results from this study revealed that bringing complex natural communities into the mesocosm caused a substantial loss of individuals and species, mainly due to translocation and disturbance effects. This important insight into the effects of bringing community assemblages into the mesocosm confirms that even with a loss of diversity, the L4 community maintained functionality and was resilient to alterations in DO. This suggests that the L4 benthic community does not depend on any one specific species for the provision of important ecosystem processes, resulting in considerable functional resilience within the L4 system. However, vulnerability to benthic systems may increase if functionality is dominated by species such as A. filiformis. Consequently, moderate hypoxia may not immediately affect benthic communities in terms of structure and diversity, but the physiological effects on individuals, especially to reproductive development, may cause alterations in the quality and quantity of planktonic propagules supplied by benthic species to the pelagic environment. This could affect benthic community diversity and functionality in the long term if repeated hypoxic events occur.