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Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

In an effort to provide wastewater treatment operators with the capability to be proactive in assessing and solving deflocculation events, this study has tested the components of a biosensor to predict deflocculation and investigated the mechanistic cause of deflocculation relating to chlorination of activated sludge cultures. In order to effectively manage upset events, it is necessary to know the source of an upset and the causative mechanism that the source initiates. The Glutathione-gated potassium efflux (GGKE)induced activated sludge deflocculation biosensor incorporates novel microtechnology with a whole cell biological element to predict deflocculation from electrophilic sources. This sensor utilizes microfluidic channels to conduct influent wastewater across a biofilm of Eschericia coli K 12 and monitors the bacterial response to the influent. The bacterial response, which is efflux of K+ ion from the cytoplasm, is monitored with a fluorescence-based sensor called an optode. The components of the system satisfy the project requirements, which include minimal expense (both operation and manufacture), on-line capability and minimal maintenance. The research conducted to date demonstrates the ability of the components of the biosensor to fulfill the design requirements. The optode K+ detector successfully measured an increase in soluble K+ following the exposure of E. coli K-12 to the electrophile N ethyl malemide. The manufacture of the microfluidic device has been completed and the device has demonstrated the ability to conduct influent under negative pressure across an established biofilm with the optode in place. The establishment of a biofilm under expected hydrodynamic conditions has also been completed. Future research efforts will include integrating the components of the biosensor into a working prototype that will be capable monitoring the reaction of bacteria to the presence of electrophilic compounds in wastewater. Sensors of this nature will provide operators with the early warning necessary to be proactive against toxic upsets rather than reactive. The knowledge needed to create a biosensor resides in the identification of bacterial response mechanisms that cause upset events in wastewater treatment facilities. The biosensor that has been developed relies on the discovery of the link between electrophile-induced GGKE and activated sludge deflocculation. Research has been concluded, which expands the role of GGKE and activated sludge deflocculation to include chlorine-induced GGKE. Through a series of laboratory-scale reactors, a relationship has been established between chlorine addition to control filamentous bulking, increased soluble K+ levels and an increase in effluent suspended solids . The results demonstrate that the addition of chlorine to control filamentous bulking may elicit the GGKE mechanism, initiating activated sludge deflocculation, similar to observations of chlorination at full-scale activated sludge wastewater treatment facilities. Establishing a mechanistic cause of deflocculation related to chlorination will permit operators to apply chlorine in a manner that may avoid deflocculation, rather than reacting to deflocculation after it has occurred. / Master of Science

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

While cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD), mechanistic insights and effective therapies are lacking. Multiple toxins accumulating as a consequence of CKD have been identified, yet the consequences for cellular crosstalk in the brain and the mechanisms underlying the associated neuronal dysfunction remain largely elusive. In the case of CKD, more than 100 uremic toxins have been identified. Renal transplantation largely reverses the cognitive impairment associated with CKD, demonstrating that cognitive impairment in CKD can be reversed. This indicates that pharmaceutical approaches to target cognitive impairment in association with CKD may be feasible. However, it is unlikely that targeting a single toxin will be sufficient to combat neuronal dysfunction associated with peripheral diseases such as CKD, given the large number of toxins involved and since the pattern of accumulating toxins varies among affected patients. Rather than identifying single toxins, identifying a common mechanism inducing neuronal dysfunction and thus impairing cognition may identify new and feasible therapeutic approaches. One commonality of peripheral diseases such as liver or renal failure is sterile inflammation. Sterile inflammation has been linked with neurodegenerative diseases and associated cognitive impairment and inflammasome activation is one hallmark of chronic pathologies in the brain. Mutations in the inflammasome component NLRP3 show clinical manifestations of cryopyrin- associated periodic syndromes (CAPS), which are characterized by skin rash, fever and joint pain. Further, abnormal and constant NLRP3 signaling has been associated with some chronic and degenerative diseases such as Alzheimer’s disease (AD), atherosclerosis, arthritis or cancer. A causative function of the NLRP3 inflammasome for neurodegenerative processes is supported by preclinical studies. These pre-clinical studies used whole body knock out mice to demonstrate that deficiencies of NLRP3, caspase-1 or the primary receptor for IL-1β, IL-1R1, protect mice from neurodegenerative processes. While providing important insights into the role of the NLRP3-inflammasome in neurodegenerative processes, these studies did not identify the relevant cell types in which the inflammasome is activated, the mechanisms underlying inflammasome activation and the consequences thereof, e.g. for intracerebral cross-talk. In addition, whether sterile inflammation triggered by the NLRP3 inflammasome impairs cognition in the setting of primarily peripheral diseases such as CKD remains unknown. To address these open questions, I used a mouse model of CKD, in which I detected NLRP3 inflammasome in brains. Interestingly, despite inflammasome activation in the brain, microglial caspase-1 deficiency did not improve cerebral inflammation and cognition in CKD mice. I identified noncanonical IL-1β maturation in microglia in CKD conditions, which was cathepsin c – caspase-8 mediated. Restoring K+ homeostasis in microglia or genetic inhibition of neuronal IL-1R1 signaling abolished CKD-induced cognitive impairment. Mechanistically, noncanonical IL-1β maturation and secretion from microglia promotes via IL-1R signaling cognitive impairment in neurons. This identifies a molecular mechanism of sterile CNS inflammation and the associated intercellular signaling pathway, which may be therapeutically amendable. Microglial K+ dyshomeostasis and noncanonical microglial IL-1β maturation may be druggable targets in some forms of cognitive impairment.:Content 2 List of abbreviations 5 Graphical abstract 8 2 Introduction 9 2.1 Chronic kidney disease and cognition 11 2.2 Microglia cells 13 2.3 The inflammasome, potassium dyshomeostasis in brain cells and thallium autometallography 15 2.4 Sterile inflammation in neurodegenerative diseases 17 3 Aims of the study 19 4 Materials and Methods 20 4.1 Reagents 20 4.2 Mice 27 4.3 CKD mouse model (5/6 nephrectomy model) 30 4.4 Evans Blue extravasation assay 32 4.5 2-photon microscopy 32 4.6 Analysis of mice 33 4.7 In vivo interventions 33 4.8 Histology and immunohistochemical analysis 34 4.9 Cell culture 34 4.10 Dextran permeability assay 35 4.11 Thallium-AMG (TlAMG), ex vivo and in vitro 36 4.12 Protein extraction and Western blotting 38 4.13 IL-1β ELISA 38 4.14 Reverse Transcriptase Polymerase Chain Reaction (RT–PCR) 38 4.15 Proximity ligation assay (PLA) 39 4.16 Behavioral analysis 39 4.17 Cathepsin c substrate assay 40 4.18 snRNA-Seq 41 4.19 Statistical Analysis 42 5 Results 43 5.1 Chronically impaired renal function leads to cognitive decline 43 5.2 Blood brain barrier (BBB) disruption in chronic kidney disease 44 5.3 Potassium dyshomeostasis in brain cells in CKD 45 5.4 CKD leads to microglia activation 46 5.5 Priming of microglia in CKD depends on potassium dyshomeostasis and its restoration improves cognition in CKD 49 5.6 TRAM34 ameliorates potassium dyshomeostasis and behavior in CKD 51 5.7 Uremia-induced cognitive impairment depends on microglia- neuron crosstalk via IL-1R1 52 5.8 Deciphering the microglial molecular pathway in CKD 56 5.9 Microglia activation in CKD is independent of NLRP3 56 5.10 Microglial IL-1β maturation occurs independently of the NLRP3-Caspase-1 inflammasome in CKD 57 5.11 The role of caspase- 8 in microglia activation in CKD 60 5.12 Lysosomal cathepsin c promotes microglia activation pivotal for caspase-8 activation 62 5.13 Broader implication of the pathway in other chronic peripheric diseases 63 5.14 Microglia inflammasome activation and IL-1β release is sufficient to induce cognitive impairment 64 5.15 Tables 66 6 Discussion 69 7 Summary 75 8 Zusammenfassung 80 9 References 86 10 Declaration about the independent preparation of the work 97 11 Presentation of own contribution 98 12 Curriculum vitae 99 13 Publications 104 14 Acknowledgments 106

This research first examined nitrification inhibition caused by different classes of industrially relevant chemicals on activated sludge and found that conventional aerobic nitrification was inhibited by single pulse inputs of every chemical tested, with 1-chloro-2,4-dinitrobenzene (oxidant) having the most severe impact, followed by alkaline pH 11, cadmium (heavy metal), cyanide, octanol (hydrophobic) and 2,4-dinitrophenol (respiratory uncoupler). Of the different chemicals tested, the oxidative and hydrophobic chemicals showed severe nitrification inhibition relative to other treatment processes and therefore deserved further investigation. For oxidative chemicals, we hypothesized that the more severe inhibition was because nitrifying bacteria lack one or more of the microbial stress response mechanisms used to mediate the toxic effect of oxidative chemicals. During these experiments, we showed that a rapid (minutes) antioxidant potassium efflux mechanism does not exist in two nitrifying bacteria, Nitrosomonas europaea and Nitrospira moscoviensis. Furthermore, we showed that another important antioxidant molecule, glutathione, was not oxidized as readily as in a non-nitrifying bacterium. Furthermore, we hypothesized that hydrophobic chemical-induced nitrification inhibition recovered more quickly because of the presence of membrane modification stress response mechanisms. While testing this hypothesis, we showed that N. europaea modified its cell membrane in response to hydrophobic chemicals using a long-term (hours) membrane modification mechanism that required the synthesis of new fatty acids, but it did not contain a short-term (minutes) response mechanism involving a cis/trans isomerase. Therefore, investigating these nitrifier stress responses showed that nitrifiers lack short-term stress responses that may be used to rapidly detect inhibition, indicating that conventional methods of detecting nitrification inhibition, like differential respirometry and nitrate generation rate (NGR), are still the fastest and easiest methods to use. Because several conventional methods exist, we also investigated differences between differential respirometry and a UV method we developed to measure NGR. During these tests, we showed that the UV NGR method provided a more reliable measure of nitrification inhibition than differential respirometry, and that the time to maximum nitrification inhibition depended on the properties of the chemical toxin, which implies that longer exposure times may be needed to accurately predict nitrification inhibition. / Ph. D.

The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria. / Ph. D.