RATIONALE: Recent studies indicate that plants can partition electron transport through alternative oxidase (AOX) and cytochrome c oxidase (COX) in response to environmental cues, thus modulating respiratory efficiency. The ¹⁸O discrimination method necessary for measuring electron partitioning in vivo, however, has been restricted to laboratory settings due to equipment constraints. Since plants grown in more natural and variable environments may not respond as predicted by laboratory experiments, I developed a new field-compatible analytical method and then applied it to three ecophysiological studies. METHODS: To address these needs, I developed a field-compatible method in which plant tissue was incubated in 12 mL septum-capped vials for 0.5 – 3 h, after which the incubation air was transferred to 3.7 mL storage vials for subsequent measurement by mass spectrometry. I also developed mathematical tools to correct for unavoidable contamination, and to detect and address curvature in the data – whether intrinsic to the respiration or due to contamination, – and to extend the usable dynamic range of the mass spectrometer. These methods were used to investigate respiratory responses (1) in canopy trees growing across a soil nutrient gradient at the Franz Josef chronosequence, New Zealand; (2) in a nutrient manipulation experiment of Griselinea littoralis; and (3) in a long-term nutrient-, temperature-, and light manipulation at Toolik, Alaska, USA. Leaf dry matter content, specific leaf area, nitrogen (N), phosphorus (P), sugars, starch, and AOX/COX protein concentrations were also measured as explanatory variables. (Leaf Cu and Fe were measured at the Franz Josef chronosequence.) RESULTS: Discrimination values computed using my methods replicated previously reported results over a range of 10 – 31‰, with precision generally better than ±0.5‰, thus demonstrating its validity as tool for measuring respiratory electron partitioning. Foliar respiration declined with site age across the soil chronosequence, increasing with leaf N levels, r² = 0.8, but electron partitioning declined with increasing N/P, r² = 0.23. AP activity was positively correlated with leaf P, Cu, and starch, r² = 0.71. In younger soils, however, declines in respiration were attributed to declines in cytochrome pathway (CP) activity, whereas across the older sites respiration declined due to a reduction in AOX pathway (AP) activity. The Griselinia nutrient-manipulation experiment partially confirmed these results: AOX protein levels were highest in N-deficient plants rather than in plants deficient in both N and P. AP activity was very low in all leaves, however, possibly due to low illumination. In support of this claim, leaves that had developed in the sun had higher AOX/COX protein ratios than those that had developed in the greenhouse. In Griselinia roots, CP activity declined by more than half in response to nutrient deficiency, whereas AP activity was unchanged. At the Arctic site, only one species changed electron partitioning in response to nutrient addition. Betula nana, the most successful adapter to improved mineral nutrition, doubled leaf CP activity without changing AP activity. Species grown in full sun at that site also had higher AOX/COX protein ratios than those that grew in enclosures. CONCLUSIONS: This is the first study of how engagement of terminal respiratory oxidases in plants responds to multiple nutrient deficiencies, both in nature and in a controlled environment. I have uncovered some intriguing relationships, including the possible importance of N/P to electron partitioning, as well as a role for Cu. The results also suggest that electron partitioning is sensitive to plant energy balance, as suggested by the low AP activities and low AOX/COX protein ratios in shaded plants. Perhaps most significantly, the AP and CP appeared to act independently of each other, rather than through a concerted “partitioning” process. In addition to their own scientific merit, these results illustrate the value of using the new field-compatible method to conduct ecophysiological investigations of plant respiratory electron partitioning on a much large scale, and under more realistic conditions, than has been previously possible.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. / Thesis / Doctor of Philosophy (PhD)

Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability.

Dans le but de maintenir leur métabolisme et leur prolifération exacerbée, les tumeurs sont dépendantes d’un apport accru en acides aminés. Afin d'optimiser cet apport, les tumeurs surexpriment certains transporteurs clés tels que l'hétérodimère multifonctionnel CD98/LAT1. CD98 (SLC3A2) agit comme co-récepteur des intégrines β et amplifie leur signalisation régulant ainsi la migration et l'adhésion cellulaire. La protéine LAT1 (SLC7A5) est quant à elle responsable du transport des acides aminés (AA) essentiels. Des études antérieures ont suggéré que la fonction CD98/intégrines du complexe est essentielle à la croissance tumorale, alors que LAT1 aurait un rôle mineur dans ce contexte. Cependant, les besoins nutritifs accrus des cellules tumorales nous ont conduit à émettre l’hypothèse contraire selon laquelle l’avantage prolifératif donné par ce complexe serait en réalité supporté par l’activité du transporteur LAT1 et non pas par l’interaction CD98/intégrine. Dans ce contexte, j’ai montré que l’invalidation génétique ou pharmacologique de LAT1 dans différentes lignées tumorales entraine une suppression totale du transport de la leucine, sodium-indépendant. Ceci entrainant une perte d’homéostasie des AA avec l’activation de la voie de stress GCN2, l’inhibition de mTORC1 et la suppression de la croissance tumorale. De plus, l’invalidation génétique de CD98 ne s’est traduite par aucun phénotype visible. Cependant, la suppression de l'activité résiduelle de LAT1 de ces cellules est suffisante pour abolir leur potentiel tumoral. Ainsi, mes résultats démontrent le rôle clé de LAT1 dans la croissance tumorale en faisant ainsi une cible thérapeutique prometteuse. / Tumours rely on external amino acids (AA) uptake to maintain their exacerbated metabolism and proliferation. To optimize AA uptake, tumors overexpress key carriers such as the multifunctional CD98/LAT 1 heterodimer. CD98 (SLC3A2) acts as a co-receptor of β integrins and enhances signaling that promotes cellular migration and invasion. LAT1 (SLC7A5) is responsible for the transport of essential AA. Previous studies have suggested that the CD98/integrin axis of the complex is essential for tumour growth, while LAT1 activity is dispensible. However, the increased nutritional requirements of tumor cells led us to hypothesize that the proliferative advantage given by this complex is in fact supported by the AA transporter activity of LAT1 and not by the CD98/integrin activity. In this context, I have shown that genetic or pharmacological invalidation of LAT1 in various tumor cell lines leads to a complete removal of the sodium-independent leucine transport. This leads to a loss of AA homeostasis with activation of the GCN2 stress pathway, inhibition of mTORC1 and supression of tumour growth. In addition, genetic invalidation of CD98 did not result in any detectable phenotype. However, inhibition of the residual activity of LAT1 in CD98 knockout cells is sufficient to abolish their tumorigenicity. Thus, my results clearly demonstrate the fundamental role of LAT1 in tumour growth and advocate the pharmacology development of LAT1 transporter inhibitors as very promising anticancer agents.

Algae have been the centre of recent research as a sustainable feedstock for fuel because of their higher oil yield in comparison to other plant sources. However, algae biofuel still performs poorly from an economic and environmental perspective due to the high reliance on freshwater and nutrients for cultivation, among other challenges. The use of wastewater has been suggested as a sustainable way of overcoming these challenges because wastewater can provide a source of water and nutrients for the algae. Moreover, the ability of the algae to remove contaminants from wastewater also enhances the total economic output from the cultivation. However, the success of this strategy still depends greatly on efficient strain selection, cultivation and harvesting. Therefore, this PhD thesis has focussed on strain isolation, characterisation, optimisation and cultivation in open pond systems. Five algae strains were isolated from wastewater treatment tanks at a municipal water treatment plant in North West England. The isolated strains were morphologically and genetically characterised as green single-celled microalgae: Chlamydomonas debaryana, Hindakia tetrachotoma, Chlorella luteoviridis, Parachlorella hussii and Desmodesmus subspicatus. An initial screening of these strains concluded that C. luteoviridis and P. hussii were outstanding in all comparisons and better than some of the strains previously reported in the literature. Further tests carried out to elucidate the underlying tolerance mechanisms possessed by these strains were based on stress tolerance and acclimation hypotheses. In the following experiments, C. luteoviridis and P. hussii were found to have higher anti-oxidant enzyme activity that helps in scavenging reactive oxygen species produced as a result of exposure to wastewater. This result provides a new argument for screening microalgae strains for wastewater cultivation on the basis of anti-oxidant activity. In addition, the two strains could grow heterotrophically and are better adapted to nutrient deficiency stress than the other three isolates. In order to understand the role of microalgae acclimation in wastewater cultivation, strains identical or equivalent to the wastewater treatment tank isolates were obtained from an algae culture collection. These strains had not been previously exposed to wastewater secondary effluent. The initial growth of these strains in wastewater secondary effluent was very poor. However, after two months of acclimation to increasing concentrations of secondary wastewater effluent, it was observed that growth, biomass and lipid productivities of most of the strains were significantly improved, although still not as high as the indigenous strains. Therefore, it was concluded that continuous acclimation is an additional factor to the successful growth of algae in wastewater. Furthermore, addition of 25% activated sludge centrate liquor to the secondary effluent was found to increase algal growth and biomass productivity significantly. Futher tests to examine the continous cultivation of C. luteoviridis and P. hussii in wastewater showed that a biomas productivity of 1.78 and 1.83 g L-1 d-1 can be achieved on a continual basis. Finally, the capability of C. luteoviridis and P. hussii for full seasonal cultivation in a 150 L open pond in a temperate climate was studied, using the optimised secondary wastewater +25% liquor medium. Each strain was capable of growth all year including in autumn and winter but with strongest growth, productivity and remediation characteristics in the summer and spring. They could maintain monoculture growth with no significant contamination or culture crash, demonstrating the robustness of these strains for wastewater cultivation in a northern European climate.

Carnivorous plants perform as both producers and consumers. Botanical carnivory has evolved in sunny, moist, nutrient-poor environments, and the primary nutrient supplied by prey is proposed to be nitrogen. There is a trade-off between carnivorous and photosynthetic structures which corresponds to degree of carnivory expression and available nutrients. This study was conducted on the hooded pitcher plant, Sarracenia minor, which is a facultative wetland plant and Florida-threatened species. Sarracenia minor is considered a specialist myrmecophage and ants characterize the majority of attracted and captured prey. Ants not only provide nutritional benefit, but also protection against herbivory. A natural population of S. minor in northeast Florida was selected to test response to prey and fertilizer nitrogen in a press-experimental design. Introduced fire ants (Solenopsis invicta) and ammonium nitrate (NH4NO3) were used as prey and fertilizer nitrogen sources, respectively. Treatments included: 1) ant addition; 2) fertilizer addition; 3) ant addition/fertilizer addition; 4) no ants/no fertilizer; 5) control. Treatments were administered biweekly and morphological characteristics and herbivory were measured monthly from April-November 2012. Results indicated no significant treatment effects on plant performance and morphological characteristics, except for a significantly greater number of flowers displayed by the nutrient-deprived group (p < .005). Herbivory by Exyra semicrocea also showed a marginally significant negative effect on the tallest pitchers per ramet. Since nitrogen is primarily stored by pitchers and allocated to new growth in the following growing season, the predictive power of this study may be limited. However, increased flowering rate in the nutrient-deprived group suggests that plants were induced to flower from nutrient stress. Also, a burn at the beginning of the study likely influenced nutrient availability and plant response to experimental treatments. In conclusion, stress may have occurred from both fire and nutrients, and S. minor showed resistance and poor response to nitrogen addition.

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Marine and coastal ecosystems are subject to diverse and increasingly intensive anthropogenic activities, making understanding cumulative effects critically important. However, accurately accounting for the cumulative effects of human impacts can be difficult, with the possibility of multiple stressors interacting and having greater impacts than expected, compounding direct and indirect effects on individuals, populations, communities and ecosystems. Assessment of multiple stressors therefore requires extensive scientific research that directly tests how single or multiple ecological components are affected by stressors, both singly and when combined, and as a consequence, cumulative effects assessments are now increasingly included in environmental assessments. Currently, there is a need to assess these at larger spatial scales, with additional research also urgently needed on the responses of ecological components, processes and functions to single and cumulative stressors. As cumulative environmental impacts could be better addressed by regional stressor effects assessments that combine methods for predicting multiple pressures on ecosystem recovery alongside degradation, this study used several separate approaches that can be used in parallel to give support for local management measures. I tested four completely different methods - a range of multi-metric indices, a food web model (Ecopath), a predictive model (Ecosim) and a Bayesian Belief Network model. Each approach was tested and compared in two shallow water estuarine systems, in Scotland and England, initially concerning the impact of nutrient enrichment and subsequent recovery and was followed by an investigation of how the addition of multiple stressors (nutrient levels, temperature and river-flow rates) would impact the future state of each system. The response to stressors was highly context dependent, varying between and within geographic locations. Overall, each of the four different approaches complemented each other and gave strong support for the need to make big reductions in the pressures and to consider trade-offs between impacting pressures. The models and tools also indicate that in order to reach an improved overall environmental state of each ecosystem, a focus on nutrient reductions are likely to be the most effective of the controls on stressors explored and that cumulative effects of the management of nutrient inputs and increased water temperatures and river-flow are likely to exist.

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