Probiotics are living organisms which exert a beneficial health effect when consumed in sufficient numbers. Consumer interest in probiotics has increased dramatically in recent years prompting an increase in production and development of functional foods. One major problem is the decreased viability of probiotic bacteria during functional food production and storage and subsequent digestion due to environmental stresses. The most common probiotic strains belong to the genus Lactobacillus or Bifidobacterium. Due to the anaerobic nature of these bacteria, they lack the required defense mechanisms for oxidative stress inherent in aerobic microorganisms. This study examined the oxidative stress responses of six strains of Bifidobacterium, which are commonly used as probiotics in functional foods.The first phase of the study investigated the innate and inducible hydrogen peroxide (H2O2) stress response of Bifidobacterium longum strains NCC2705 and D2957, Bifidobacterium longum ssp. infantis ATCC 15697, and Bifidobacterium animalis ssp. lactis strains BL-04, DSM10140 and RH-1. Strains were screened for survival at increasing concentrations of H2O2 and lethal and sublethal concentrations were determined for each. In the second phase, B. animalis ssp. lactis strains BL-04 and DSM10140 and B. longum strains NCC2705 and D2957 were treated with a sublethal H2O2 concentration and RNA samples were collected for transcriptome analysis after 5 min and either 20 or 60 min. Statistical analysis was performed to identify genes that increased or decreased in expression during H2O2 treatment compared to control cells.Results showed that survival was species and strain dependent and that strains which naturally survived higher H2O2 concentrations had a larger number of differentially expressed genes early on during H2O2 exposure. Some of the protective genetic systems that were activated during H2O2 stress are mechanisms which perform basic cellular functions under normal conditions such as deoxuynucleotide synthesis. Under stress conditions, these systems can be used to detoxify oxidative free radicals. Also a number of genes involved in sugar transport and energy production for the cell showed increased expression, which reveals the increased energy needs of the cells during oxidative stress.During testing, it was found that two B. animalis ssp. lactis strains, BL-04 and DSM10140, had differing levels of survival and gene expression during H2O2 exposure despite having almost identical genome sequences. It was determined that one possible cause of the differences was a genetic deletion in a gene that allows the cell to incorporate extracellular fatty acids into the cell membrane instead of synthesizing them.Results from this project have increased the understanding of oxidative stress responses in bifidobacteria and highlighted possible methods to increase bacterial survival during food manufacture, storage, and human digestion.

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

The E. coli ArsA is involved in arsenic detoxification but the role of yArsA (ArsA homologue of Saccharomyces cerevisiae, encoded by YDL100c ORF) in yeast is still undefined. Disruption of YDL100c ORF is not lethal but the disrupted strain (KO) shows decreased thermotolerance. To study the role of yArsA in thermotolerance, wild type (WT) and KO were grown at 25¢Jand 37¢J, and assayed for the intracellular levels of trehalose accumulation and molecular oxidation, and the biosynthesis of heat shock proteins. The results show that molecular oxidation is higher and trehalose accumulation is lower in KO compared with WT grown at 37¢J, suggesting that increased ROS and decreased trehalose content are the cause of cell death. Further analysis of the expression of ROS defense mechanisms show that there is no significant difference in TSL1 and SOD1 expression in WT and KO grown at 25¢J or 37¢J but the CTT1 expression in KO was much less than WT grown at 37¢J. These observations are consistent with the assays of enzymatic activity of catalase and antioxidant GSH contents. Loss of catalase activity, decreased trehalose contents and Hsp104p expression suggest a deficiency in activation of general but not specific stress response in KO when grown at 37¢J. Therefore, yArsA was involved in signaling the general stress response in stress tolerance network.

The causative agent of malignant tertian malaria, Plasmodium falciparum undergoes an arrested growth phenotype of its erythrocytic stage when under drug-stress. Recent artemisinin treatment failures seem to be indicative of such induction followed by recrudescence rather than actual therapeutic failure. Likewise, P. vivax hypnozoites are the prototypic dormants and the latent infections for which they are responsible prove most difficult to treat. Dihydroartemisinin, an artemisinin-derivative, can be used to exploit this mechanism by inducing a dormant state in ring-stage P. falciparum parasites and in turn, their recovery may be used as a screening period for compounds that inhibit or foster growth. Specifically, parasites stably transfected with luciferase were used to quantitatively observe growth (or lack thereof) response of parasites to the phytohormone gibberellic acid and the herbicide, fluridone. Using their behavior as comparative controls, the Medicines for Malaria Venture (MMV) Malaria Box was screened for similar activity. The most active compound, 1,2,3,4-tetrahydroacridin-9-ol a quinoline-derivate caused cells to wake even earlier than expected. Since quinine and other such drugs have historically been most effective in treating malaria, it seems appropriate that such a finding was made. Following this the MMV Box was screened again against uninduced 3D7 parasites to determine if any were capable of causing a dormant response under the hypothesis that such a reaction is a defensive adaptation of P. falciparum. Four compounds were found to be active of which two appear to be inducing dormancy in the second cycle rather than the first akin to DHA. These quiescent periods also appear to be shorter indicating that the latter is more efficient. It is possible that given the length of interaction with artemisinin, P. falciparum is more adept to respond to its derivatives likewise the mechanism of action may be different enough to change the nature of the response.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.

abstract: Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background. / Dissertation/Thesis / Ph.D. Microbiology 2010

Protein phosphorylation is an essential regulatory mechanism employed by many key pathways in the eukaryotic cell. This thesis explored two examples of protein phosphorylation, one in translation and one in cell cycle regulation. TRNT1 is the RNA polymerase that adds the 3’ CCA nucleotides to all tRNA and is required for aminoacylation, tRNA quality control, and protection of the translation machinery (Aebi et al., 1990; Vörtler & Mörl, 2010; Wellner et al., 2018). Mutations in TRNT1 cause SIFD, Sideroblastic Anemia With B-Cell Immunodeficiency, Periodic Fevers, And Developmental Delay, a rare mitochondrial disease (Chakraborty et al., 2014). In addition to oxidative phosphorylation defects previously reported (Liwak-Muir et al., 2016; Sasarman et al., 2012; Wedatilake et al., 2016), I show that defects in TRNT1/Cca1 induce the eIF2α-P translation stress response to slow cytoplasmic translation but is only beneficial when mitochondrial translation is required. This data suggests that impairment of TRNT1/Cca1 function results in an imbalance of translation homeostasis and that altering the balance between mitochondrial and cytoplasmic translation may be an effective therapy for SIFD. In another example of protein phosphorylation regulation, Cdc25-dependent dephosphorylation of Cyclin dependent kinase (Cdk1) is a universally conserved mechanism regulating mitotic entry. In budding yeast, Cdk1 is dephosphorylated when the cdc25 homologue, MIH1, is deleted, indicating an additional phosphatase functions redundantly with Mih1. I identified two phosphatases, Ptp1 and PP2ARts1, that function in conjunction with Mih1. This redundancy suggests novel mechanisms for how cells may activate pools of Cdk1 at different times and in different cellular locations.

Proteases play a crucial role in plant defence mechanisms as well as acclimation to changing metabolic demands and environmental cues. Proteases regulate the development of a plant from germination through to senescence and plant death. In this thesis the role of proteases and their inhibitors in plant response to cold stress and CO2 enrichment were investigated. The activity and inhibition of cysteine proteases (CP), as well as degradation of their potential target proteins was investigated in transgenic tobacco plants expressing the rice cystatin, OC-I. Expression of OC-I caused a longer life span; delayed senescence; significant decrease in in vitro CP activity; a concurrent increase in protein content; and protection from chilling-induced decreases in photosynthesis. An initial proteomics study identified altered abundance of a cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase and two RuBisCO activase isoforms in OC-I expressing leaves. Immunogold labelling studies revealed that RuBisCO and OC-I is present in RuBisCO vesicular bodies (RVB) that appear to be important in RuBisCO degradation in leaves under optimal and stress conditions. Plants need to respond quickly to changes in the environment that cause changes in the demand for photosynthesis. In this study the effect of CO2 enrichment on photosynthesis-related genes and novel proteases and protease inhibitors regulated by CO2 enrichment and/or development, was investigated. Maize plants grown to maturity with CO2 enrichment showed significant changes in leaf chlorophyll and protein content, increased epidermal cell size, and decreased epidermal cell density. An increased stomatal index in leaves grown at high-CO2 indicates that leaves adjust their stomatal densities through changes in epidermal cell numbers rather than stomatal numbers. Photosynthesis and carbohydrate metabolism were not significantly affected. Developmental stage affected over 3000 transcripts between leaf ranks 3 and 12, while 142 and 90 transcripts were modified by high CO2 in the same leaf ranks respectively. Only 18 transcripts were affected by CO2 enrichment exclusively. Particularly, two novel CO2 -modulated serine protease inhibitors modulated by both sugars and pro-oxidants, were identified. Growth with high CO2 decreased oxidative damage to leaf proteins. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.