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Host Nectin-1 Is Required for Efficient Chlamydia Trachomatis Serovar E DevelopmentHall, Jennifer V., Sun, Jingru, Slade, Jessica, Kintner, Jennifer, Bambino, Marissa, Whittimore, Judy, Schoborg, Robert V. 01 January 2014 (has links)
Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.
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Molekularne osnove odgovora medonosne pčele (Apis mellifera, L.) nastres izazvan jonima teških metala / Molecular basis of honey bee (Apis mellifera, L.) response to heavy metal stressNikolić Tatjana 07 July 2017 (has links)
<p>Istraživanja u ovoj doktorskoj disertaciji su bila usmerena ka razumevanju<br />molekularnih mehanizama koji se aktiviraju kod medonosne pčele (<em>Apis mellifera, L</em>.) kao odgovor na stres izazvan jonima teških metala, što zbog važnosti medonosne pčele kao oprašivača ima poseban značaj i očekuje se da će doprineti očuvanju ove vrste. Istraživanja su bila podeljena u tri faze. U prvoj fazi za analize su korišćene populacije pčela sa lokaliteta sa različitim antropološkim uticajem i izmerena je koncentracija metala u pčelama, pergi i medu, kao i relativna genska ekspresija i aktivnost antioksidativnih enzima. U drugoj fazi pčele su u kontrolisanim laboratorijskim uslovima bile izložene subletalnim dozama jona teških metala (bakra, kadmijuma i olova), nakon čega su izmereni parametri koji ukazuju na redoks status i nivo oksidativnog stresa. Rezultati prve dve faze su pokazali da se ekspresija gena i aktivnost antioksidativnih enzima (superoksid dismutaze, katalaze i glutation <em> S</em>-transferaze) razlikuje u zavisnosti od stepena urbanizacije i industrijalizacije, dok je izlaganje bakru i kadmijumu u kontrolisanim uslovima u trajanju od 48 h dovelo samo do promene u ekspresiji gena i u većini slučajeva ekspresija je bila dozno zavisna od koncentracije metala. Olovo je uzrokovalo promene u koncentraciji glutationa i sulfhidrilnih grupa proteina, što govori o tome da helacija olova može da bude prvi mehanizam odbrane od toksičnih efekata ovog metala. U trećoj fazi bioinformatičkom analizom je pronađen metalotionein medonosne pčele i ispitana je njegova funkcija u zaštiti od toksičnih efekata jona teških metala. Utvrđeno je da pčele poseduju jedan gen za metalotionein, koji kodira mali protein sa regionima bogatim cisteinom za koje mogu da se vežu joni metala. Indukcija genske ekspresije metalotioneina medonosne pčele nakon izlaganja metalima i povećana tolerancija bakterija koje ekspresuju rekombinantni metalotionein na metale je potvrdila da metalotionein medonosne pčele ima ulogu u homeostazi bioelemenata i detoksikaciji potencijalno toksičnih metala. Dobijeni rezultati predstavljaju osnovu za buduća istraživanja uticaja jona teških metala na medonosnu pčelu i predstavljaju važan korak u sveobuhvatnoj proceni uticaja stresogenih faktora iz životne sredine na pčele.</p> / <p>Research in this doctoral thesis focuses on understanding the molecular mechanisms activated in the honey bee (Apis mellifera L.) as a response to stress caused by exposure to heavy metal ions. Because of the importance of honeybees as pollinators, this has special significance and is expected to contribute to the conservation of this species. Studies have been divided into three phases. In the first phase, bee populations from three localities under different anthropological influence were used and the concentrations of metals in the bees, honey and bee bread (perga), as well as relative gene expression and activity of antioxidant enzymes were measured. In the second phase, bees were exposed to sublethal doses of heavy metal ions (copper, cadmium and lead) under controlled laboratory conditions, after which parameters that indicate redox status and oxidative stress were determined. The results of the first two phases showed that gene expression and activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione S-transferase) varies depending on the degree of urbanization and industrialization, while exposure to copper and cadmium in controlled conditions for 48 h resulted only in a change in gene expression in the majority of cases, and the expression was dose-dependent on the concentration of the metal. Lead has caused changes in the concentration of glutathione and sulfhydryl groups of proteins, which indicates that chelation may be the first defense mechanism against the toxic effects of this metal. In the third stage, honeybee metallothionein was identified by bioinformatic analysis and its function in protection against the toxic effects of heavy metal ions was examined. It has been found that honeybees have one metallothionein gene, which encodes a small protein with cysteine-rich regions that may bind metal ions. The induction of metallothionein gene expression after exposure of honeybees to metals and increased tolerance of bacteria that express recombinant metallothionein confirmed that this protein plays a role in the homeostasis of bioelements and detoxification of potentially toxic metals. These results form the basis for future research on the impact of heavy metal pollution on the honey bee and represent an important step in the comprehensive assessment of the impact of stress factors from the environment on honey bees.</p>
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The Role of the S. cerevisiae Sco2p and Its Homologues in Antioxidant Defense MechanismsEkim Kocabey, Aslihan 14 September 2018 (has links)
The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis.
Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants.
Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function.
The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents
List of figures x
List of tables xii
Abbreviations xiii
1 Introduction 1
1.1 ROS production 1
1.2 Oxidative stress 2
1.3 Antioxidant response 3
1.4 The thioredoxin fold: From structure to function 6
1.5 Sco proteins 7
1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8
1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9
1.6 Background studies 10
1.7 Using yeast as a model 11
1.7.1 Cross-species complementation studies 11
1.7.2 Yeast model for human mitochondria studies 12
1.8 Aim of the study 12
2 Materials & Methods 14
2.1 Materials 14
2.1.1 Chemicals and Reagents 14
2.1.2 Equipments 16
2.1.3 Kits 17
2.1.4 Antibodies 18
2.1.5 Plasmid 18
2.1.6 Primers 19
2.1.7 S. cerevisiae strains 22
2.1.8 Media 22
2.2 Methods 24
2.2.1 Cultivation of S. cerevisiae cells 24
2.2.1.1 Culture conditions 24
2.2.1.2 Preparation of glycerol stocks 24
2.2.2 Molecular Biology Methods 24
2.2.2.1 S. cerevisiae genomic DNA isolation 24
2.2.2.2 RNA isolation 25
2.2.2.2a Cultured mammalian cells (HEK293) 25
2.2.2.2b Drosophila melanogaster 25
2.2.2.3 RNA purity and concentration determination 25
2.2.2.4 Reverse transcription 25
2.2.2.5 Polymerase chain reaction 25
2.2.2.5a Standard PCR 25
2.2.2.5b Overhang PCR 26
2.2.2.5c Overlap extension PCR 27
2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27
2.2.2.6 DNA agarose gel electrophoresis 28
2.2.2.7 DNA gel extraction and clean-up 29
2.2.2.8 DNA sequencing 29
2.2.2.9 Southern blotting 29
2.2.2.9a DNA preparation 29
2.2.2.9b Blotting 30
2.2.2.9c Preparation of a DIG-labelled probe 30
2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30
2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31
2.2.2.10 Yeast transformation 32
2.2.2.11 Growth assay 32
2.2.3 Protein methods 33
2.2.3.1 Isolation of crude mitochondria from yeast 33
2.2.3.2 SDS-PAGE 33
2.2.3.3 Protein transfer 34
2.2.3.4 Colloidal Coomassie gel staining 34
2.2.3.5 Protein detection 35
2.2.3.6 Stripping the membrane and reprobing 35
2.2.4 Biochemical methods 35
2.2.4.1 Methylene Blue staining 36
2.2.4.2 Quantification of ROS 36
2.2.4.2a Amplex Red staining 36
2.2.4.2b Lipid peroxidation assay 36
2.2.5 Bioinformatics 37
2.2.6 Statistical Analysis 37
3 Results 40
3.1 Selection of homologues by bioinformatic analysis 40
3.2 Generation of recombinant strains 42
3.3 Confirmation of site-specific integration by check PCR 44
3.4 Verification of single site integration by Southern Blotting 44
3.5 Analysis of the functional homology between selected homologues and ySCO2 45
3.5.1 Complementation assay in solid media 45
3.5.2 Complementation assay in liquid media 47
3.6 Determination of cell viability 48
3.7 Quantification of ROS 51
3.7.1 Quantification of extracellular H2O2 51
3.7.2 Quantification of lipid peroxidation 53
3.8 Investigation of the expression and subcellular localization of homologues 55
3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58
3.10 Mutational analysis of ySCO2 60
3.11 Identification of functionally important residues 61
3.12 Prediction of salt bridges 65
3.13 Alanine mutagenesis 66
4 Discussion 68
4.1 Functional homology between the selected homologues and ySCO2 68
4.1.1 A. thaliana homologues, HCC1 & HCC2 68
4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69
4.1.3 D. melanogaster homologue, SCOX 70
4.1.4 Yeast homologues, K07152 & SpSCO1 70
4.2 The localization and expression pattern of homologues 71
4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72
4.4 Mutational analysis of ySCO2 73
4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74
4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78
5 Summary 81
6 References 84
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Change of Physical Context Impairs Cardiovascular Habituation to StressPalmer, Kevin M. 01 January 2008 (has links)
The present study examined whether cardiovascular habituation to stress is affected by a change in the physical context in which a stressor is encountered. Twenty-five undergraduate students at the University of Central Florida, Palm Bay Campus, were exposed to 4 trials of a stressor consisting of mental arithmetic while under evaluative observation. It was hypothesized that if participants experienced a change in the physical context in which stress was experienced on the final trial, they would demonstrate impaired habituation to stress as indicated by measures of heart rate and blood pressure. Physical context was manipulated by either asking participants to move to another room upon the final exposure to the stressor or to remain in the same room in which they were initially exposed to the stressor for the final exposure. Participants were randomly assigned to one of 2 conditions, the Stable Room Condition (N = 10) or Novel Room condition (N = 15 ). Participants in the Stable Room Condition remained in the same physical context, or same room, throughout all trials and displayed habituation of systolic .blood pressure, diastolic blood pressure, and heart rate. Participants in the Novel Room condition were exposed to the same stressors, but were moved to a different physical context, or new room upon the final trial. The results demonstrated that participants in the novel room condition displayed significantly impaired habituation on measures on systolic blood pressure (p < .001) and diastolic blood pressure (p < .001). However, no significant difference in heart rate was observed between groups. These results indicate that a simple change in the physical context in which stress exposure occurs impairs cardiovascular habituation to stress. Implications and directions for future research are discussed.
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Characterizing the role of the bifunctional glutamyl-prolyl-tRNA synthetase in humandiseasesJin, Danni January 2021 (has links)
No description available.
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Stress Related Emissions of Norway Spruce PlantsPettersson, Marie January 2007 (has links)
The interactions between plants and insects are mediated by volatile molecules. Plants respond to stress by biosynthesis of chemical substances which can deter invading insects or pathogens. Some of these substances are volatile and are emitted to the surroundings and may attract or repel insects. Information about the susceptibility of individual plants to infestation, their volatile emissions and chemical defence is of interest, for example in selecting plants for tree breeding programs. This research was focused on finding volatile chemical markers of resistance in Norway spruce plants that do influence insects associated to conifers. Collection of headspace volatiles by SPME followed by separation and identification with GC-MS is effective in investigating biological systems with a minimum of disturbance. This method has here been used to investigate Norway spruce plants of different ages and stress conditions as well as trapping semiochemicals like nepetalactone emitted by the spruce shoot aphids. It was even possible to analyse the emission of single needles in vivo and obtain a chemical pattern of the site of the stress reaction. Seedlings of different ages showed differences in chemical composition of emitted volatiles, with the pine weevil repellent (S)-(-)-limonene as one of the main compounds. Wounded phloem of conventional plants emitted high amounts of monoterpenes while the phloem of mini plants emitted (3Z)-hexenal and (3Z)-hexen-1-ol. Norway spruce plants did respond to different stress elicitors with similar response, regardless of their genetic origin. The emissions from stressed Norway spruce plants mainly consist of (E)-β-farnesene, (E,E)-α-farnesene, (E)-α-bisabolene, (R)-(-)-linalool and methyl salicylate. Emissions from live spruce shoot aphids were detected during autumn periods, and a method to separate and identify the four diastereomers of nepetalactone by GC-MS and characteristic m/z-fragments was accomplished.
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Surgical stress response in patients with perioperative statin and/or beta-blocker treatment during colon cancer surgeryLindgren, Arvid January 2022 (has links)
Background: Surgical stress during resection surgery for colon cancer has previously been shown to be associated with adverse postoperative outcomes. Statin and beta-blocker treatment have been shown to lower postoperative complications and mortality, and been hypothesized to reduce the surgical stress response, although this correlation has not been studied clinically. Aim: To investigate whether perioperative beta-blocker and/or statin treatment reduce the postoperative C-reactive protein (CRP) response. Material and methods: All patients who underwent right sided hemicolectomy or sigmoid resection for cancer at Örebro University Hospital during 2012-2017 were included in this study. Initially, any treatment with statins, beta-blockers or both were compared to those with no treatment. After initial analyses, four treatment groups were compared regarding postoperative CRP response, namely no treatment, statin, beta-blocker, and combination treatment. Comparisons regarding complications were also performed for the four groups. Results: A total of 260 patients were included in this study. The no treatment group had a lower peak postoperative CRP than the treatment group, when comparing any treatment versus no treatment. There were no significant differences in postoperative CRP within the four treatment groups. There was no significant difference in complication rate between any of the treatment groups when compared to no treatment. Conclusion: Treatment with statin or beta-blocker therapy does not reduce the postoperative CRP response. A combination of both treatments demonstrated a trend towards a reduction regarding postoperative CRP response compared with the two treatments individually assessed. Larger studies are needed to verify the results of this study.
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The Effects of Reduced Mrpl54 Expression on Mouse Lifespan, Metabolic Health Span, and Skeletal Muscle AgingReid, Kimberly 20 February 2024 (has links)
With age comes a decline in the dynamic regulation of a balanced and functional mitochondrial proteome (proteostasis) that leads to an increase in oxidative stress and macromolecule damage, with a decline in ATP production. Compromised protein networks and reduced available energy leaves an organism susceptible to accelerated aging and the onset of age-related disease. Since mitochondrial respiratory complexes are composed of protein subunits from both mitochondria and nuclear genomes, their assembly relies on the coordination of mitochondrial and cytoplasmic translation machinery. Disruption of mitochondrial translation generates an imbalance in the ratio of mitochondrial (mtDNA) to nuclear DNA (nDNA) encoded proteins, which is called a mitonuclear protein imbalance. In response to the protein imbalance, a retrograde stress signal is sent from the mitochondria to the nucleus, invoking the mitochondrial unfolded protein response (UPRᵐᵗ) to resolve the mitoproteostatic stress. In a young healthy cell, the UPRᵐᵗ upregulates protein folding chaperones and proteases to resolve the consequences of a mitonuclear protein imbalance. In the early stages of aging, the UPRᵐᵗ appears to be upregulated in response to age-related mitochondrial proteostatic stress. In aged senescent cells however, the UPRᵐᵗ response is blunted. There is cross-species evidence that induction of the UPRᵐᵗ through moderate-intensity exercise or through genetic disruption of the mitochondrial translation machinery will act as a hormetic - resulting in health benefits in the long term. Caenorhabditis elegans longevity models demonstrate that a reduction in mitochondrial ribosomal protein (Mrp) gene expression or disturbed mitochondrial translation will function as a hormetic. The disruption of the mitochondrial ribosome leads to a mitonuclear protein imbalance, invokes the nematode UPRᵐᵗ, which then robustly extends C. elegans lifespan and health span. To determine whether the hormetic effects of mild mitochondrial ribosome disruption can be recapitulated in a mammalian model, this thesis tests a C57/BL6/NTaconic mouse model altered in the germline to have reduced Mrpl54 expression through heterozygous mutation. Mice were metabolically tested at ages 6-, 18-, and 24-months and followed through their natural lifespan to determine whether reduction in the expression of a critical Mrp (Mrpl54) impacts lifespan or metabolic health span. While Mrpl54 mRNA expression was ~50% of wildtype in all Mrpl54⁺ᐟ⁻ tissues tested, there were no differences observed in metabolic health with age or lifespan in either male or female mice. Cultured Mrpl54⁺ᐟ⁻ primary myoblasts had lower absolute levels of nDNA- and mtDNA-encoded respiratory complex subunits relative to wildtype; however, the ratio between nDNA- and mtDNA-encoded protein subunits remained like wildtype. Further testing of the model revealed that Mrpl54⁺ᐟ⁻ males had weaker grip strength by age 12-months, which was also found in the data from multiple heterozygous Mrp (Mrp⁺ᐟ⁻) mouse models available at the International Mouse Phenotyping Consortium. 12-month-old Mrpl54⁺ᐟ⁻ males displayed reduced tetanic force and better fatigue recovery in ex vivo skeletal muscles, and the transmission electron micrographs of skeletal muscle sarcomeres revealed an early aging phenotype. Unlike the C. elegans reduced Mrp longevity model, reduced expression of a critical Mrp did not result in lifespan or metabolic health span benefits in a mouse model. In contrast, the Mrpl54⁺ᐟ⁻ male model showed evidence of premature skeletal muscle aging. While the results of this research do not support the role of Mrpl54 reduced expression in mammalian lifespan or health span extension, they do point to a premature aging phenotype for certain muscle parameters that may be relevant to people living with heterozygous mitochondrial protein mutations. Typically, these individuals are regarded as carriers and free of phenotype associated with their mitochondrial protein mutation. The results in this thesis suggest that those with a heterozygous mitochondrial protein gene mutation may manifest a phenotype as they grow older and are less resilient to external or internal challenges.
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Understanding <i>Campylobacter jejuni</i> colonization and stress survival mechanisms: Role of Transducer Like Proteins (Tlps) and Polyphosphate kinases (PPKs)Chandrashekhar, Kshipra January 2014 (has links)
No description available.
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Elucidating the Role of Toxin-Induced Microbial Stress Responses in Biological Wastewater Treatment Process UpsetBott, Charles Briddell 16 April 2001 (has links)
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.
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