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Effects of anxiolytic compounds of diverse chemical classes (chlordiazepoxide, buspirone, ritanserin, propranolol and BRL46470) on the behaviour of mice were examined in several animal models of anxiety. These models included social interaction by male mice in the animal's home cage and an unfamiliar neutral cage, the light/dark box and responsiveness of the mice to calls from predatory and non-predatory birds. Effects of anxiolytics were also examined in mice in the timid female paradigm, and on the behaviour of marmosets when confronted with their unfamiliar conspecifics. Effects of anxiolytics in mice were compared with those of quinpirole, given at anxiogenic dose levels. The results showed that during social interaction, anxiolytic compounds released behaviour of animals that normally was suppressed by social and environmental constraints. In male mice, anxiolytics also increased exploratory digging and in some cases enhanced aggression. Quinpirole, however, in these models, decreased their social investigation, non-social activities as well as locomotor activities while increasing their flight behaviour. In the present studies, the light/dark box model was found to be sensitive to chlordiazepoxide and several other anxiolytics after acute administration. Effects of three classical antidepressants (imipramine, phenelzine, mianserin) on social interaction in male mice were also screened, using similar ethological techniques. It was found that the effects of antidepressants following different time courses were different from the effects of anxiolytics. When given acutely, antidepressants induced a range of diverse changes to behaviour which included anxiogenic-like effects, such as decrease of aggressive behaviour and social investigation. After subchronic administration, antidepressants exhibited anxiolytic-like effects in that they increased social investigation and sometimes increased aggression. Enhancement of exploratory digging was found only by subchronic administration of one of the antidepressants tested. Commonalities in the behavioural effects induced by anxiolytics and antidepressants when given by long-term administration indicate that they may be acting at common pathways in the central nervous system. They may act to suppress functioning of the behavioural inhibition system and also influence the hippocampal-5-HT defence reactions. The dissimilarities of behavioural effects produced by anxiolytics and antidepressants suggest that these drugs may alter defensive responding via different neurochemical routes

Loss of the ssp1 protein kinase (CAMKK) gene results in stress sensitivity, cell elongation, slow growth and in some cases cell cycle arrest. In order to identify new components of the ssp1 stress response pathway, a transposon mediated suppressor screen was used to identify loss of function suppressors of a Schizosaccharomyces pombe ssp1 gene disruption. The Musca domestica Hermes transposon was used to randomly insert the KanMx6 selectable marker in the genome. The selection was for Hermes insertions which rescued the G2 cell cycle arrest phenotype of ssp1- when grown at pH 3.5 and 36 C. Second site mutations that rescued the cell cycle arrest and allowed for colony formation were identified. In total 121 mutant strains with elongated morphology but capable of colony formation at pH 3.5 and 36 C were isolated and 22 insertion sites were identified by inverse PCR and sequencing. Genes for a transcriptional suppressor, scr1 (SPBC1D7.02c ), a spermidine transporter (SPCC569.05c ), cyp9 cyclophilin 9 (SPCC553.04), complexed with cdc5 (cwf4) (SPBC31F10.11c), ptr8 (SPAC17A5.06), (SPBC1921.07c), and set7 (SPCC297.04c) were identified as second site loss of function suppressors of the ssp1 deletion. Identifying these genes and their phenotype in conjunction with loss of ssp1, substantially improves our understanding of the Ssp1 molecular pathway in cell cycle control and cell stress response / Thesis (Master, Biology) -- Queen's University, 2014-01-27 15:55:29.75

The work presented in this thesis describes the characterisation of the L-carnitine transporter, OpuC, belonging to the binding protein dependent ABC transporter superfamily. The transporter is encoded on a four gene operon, <i>opuCABCD</i>. The physiological study of two <i>opuC</i> mutants have revealed that this operon encodes the principal carnitine transport system in <i>L. monocytogenes</i>, and that the resulting transporter is specific for carnitine and not the related solute betaine. Usually the activity of this transporter is subject to negative regulation during growth in the presence of peptone. An <i>opuCA</i> deletion mutant retained the ability to utilise carnitine as an osmoprotectant at high concentrations (1 mM), and accumulated a cytoplasmic carnitine pool comparable to the wild-type, suggesting that a second low affinity carnitine transport system must exist in <i>L. monocytogenes</i>. Measurement of carnitine uptakes rates in the presence of 100 mM and 1 mM carnitine revealed that the rate of carnitine uptake in the <i>DopuCA </i>mutant was dependent on the carnitine concentration, confirming the low affinity of this unidentified system for carnitine. The stress inducible sigma factor, s^B, is predicted to play a role in regulating the <i>Listerial </i>osmotic stress response. Studies utilising a <i>sigB</i> deletion mutant revealed that s^B is required for the utilisation of carnitine as an osmoprotectant, by regulating the transcription of the <i>opuC</i> operon in response to hyperosmotic stress. Betaine accumulation is reduced in a strain lacking s^B, in particular Na⁺ dependent betaine transport, although transcription of neither betaine transport systems, <i>gbu </i>and <i>beiL</i>, appear affected by the s^B might play a post-transcriptional regulatory role in betaine accumulation.

The transcription factors Msn2p and Msn4p are major components of the Saccharomyces cerevisiae environmental stress response. Their transient activation/deactivation is dependent on a regulatory network centering on nucleocytoplasmic shuttling but also includes a range of other mechanisms. The acetyltransferase complex, NuA4 has been implicated in repression of Msn2p yet the mechanism is largely unknown. Gds1p is an uncharacterized yeast protein identified in a recent study as a physical interactor and acetylation target of NuA4. Gds1 protein level is dependant both on NuA4 and environmental stress and our analysis shows it to be involved in the nuclear exclusion of Msn2p in the absence of stress. Unstressed cells lacking GDS1 exhibit increased nuclear accumulation Msn2p and an increase in transcription of the stress reporter gene, HSP12. My work supports a model in which Gds1 and NuA4 can work independently to inhibit the Msn2/4 dependant yeast stress response in the absence of stress.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13852号 / 工博第2956号 / 新制||工||1436(附属図書館) / 26068 / UT51-2008-C768 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第1項該当

The maintenance of protein homeostasis is vital for all cells, but it is of utmost importance in post-mitotic cells, such as neurons that cannot dilute aggregates by cell division. Dysregulation of the proteostasis network can lead to neurodegenerative disorders such as Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS), and prion diseases. The small molecule Sephin1 is a promising lead against proteostasis disruption, but its mechanism of action is uncertain. We assessed the therapeutic efficacy of Sephin1 in an established PD mouse model. Our laboratory has recently characterized a mouse expressing via bacterial artificial chromosome (BAC) the human LRRK2 G2019S protein, a variant linked to PD. Our data show that Sephin1 treatment rescues the motor deficit observed in BAC human-G2019S mice. Our experimental evidence shows that Sephin1 binds the monomeric globular actin (G-actin) in cell-free assays. By combining PAL chemistry to MS/MS analysis we identified the putative Sephin1 binding site on actin. In vitro, Sephin1 drives actin misfolding, and eventually, its precipitation. Upon Sephin1 treatment in HeLa cells, we visualized actin clusters localized to the lysosomes. This event at the lysosome impairs the normal autophagic flux. At the same time, Sephin1 induces the inactivation of the mammalian target of rapamycin (mTORC1), thus allowing the nuclear translocation of the transcription Factor EB (TFEB) and the expression of TFEB-direct target genes, on the longer term. In parallel, Sephin1 elicits the phosphorylation of the α subunit of the Eukaryotic Initiation Factor 2 (eIF2) and the ER-stress independent expression of the C/EBP homologous protein (CHOP). CHOP is a transcription factor that contributes to the integrated stress response as well as to autophagy. As such, Sephin1 triggers the activation of two main players in the autophagic response, TFEB and CHOP. Accordingly, we reported that, after the initial impairment, Sephin1 stimulates autophagy. Taken together, our results reveal a novel Sephin1 molecular mechanism in which lysosomal stress may regulate autophagy via mTORC1-TFEB complemented with the eIF2α signalling pathway. Although several questions remain to be answered, Sephin1, which successfully completed the phase I clinical trial for ALS and Charcot–Marie–Tooth disease, represents a promising therapeutic strategy that targets autophagy to regulate the homeostatic balance of proteins in neurodegenerative diseases.

Nitrification is a significant concern for drinking water systems employing chloramines for secondary disinfection. Utilities have implemented a range of disinfection strategies that have varying levels of effectiveness in the prevention and control of nitrification events, including optimizing the chlorine-to-ammonia ratio, maintaining chloramine residual throughout the distribution system, controlling pH, and temporal switching to free chlorination. Annual or semi-annual application of free chlorination is practiced by 23% of chloraminating systems on a temporary basis as a preventative measure, even though it has the undesirable consequences of temporarily increasing disinfection byproducts, facilitating coliform detachment, and altering water taste and odor. Although temporal free chlorination and other nitrification control methods have been widely studied in the field and in pilot-scale systems, very little is known about the stress responses of nitrifying bacteria to different disinfection strategies and the role physiological state plays in the resistance to disinfection. It is well known that many commonly studied bacteria, such as Escherichia coli, are able to better resist disinfection by free chlorine and chloramines under nutrient limitation through regulation of stress response genes that encode for DNA protection and enzymes that mediate reactive oxygen species. We compared the genomes of E. coli and the ammonia-oxidizing bacterium Nitrosomonas europaea, and found that many of the known stress response mechanisms and genes present in E. coli are absent in N. europaea or not controlled by the same mechanisms specific to bacterial growth state. These genetic differences present a general susceptibility of N. europaea to disinfection by chlorine compounds. Using an experimental approach, we tested the hypothesis that N. europaea does not develop increased resistance to free chlorine and monochloramine during starvation to the same degree as E. coli. In addition, N. europaea cells were challenged with sequential treatments of monochloramine and hypochlorous acid to mimic the disinfectant switch employed by drinking water utilities. Indicators of activity (specific nitrite generation rate) and viability (LIVE/DEAD® BacLight© membrane-integrity based assay) were measured to determine short-term effectiveness of disinfection and recovery of cells over a twelve day monitoring period. The results of disinfectant challenge experiments reinforce the hypothesis, indicating that the response of N. europaea to either disinfectant does not significantly change during the transition from exponential phase to stationary phase. Exponentially growing N. europaea cells showed greater susceptibility to hypochlorous acid and monochloramine than stationary phase E. coli cells, but had increased resistance compared with exponential phase E. coli cells. Following incubation with monochloramine, N. europaea showed increased sensitivity to subsequent treatment with hypochlorous acid. Complete loss of ammonia-oxidation activity was observed in cells immediately following treatment with hypochlorous acid, monochloramine, or a combination of both disinfectants. Replenishing ammonia and nutrients did not invoke recovery of cells, as detected in activity measurements during the twelve day monitoring period. The results provide evidence for the effectiveness of both free chlorine and chloramines in the inhibition of growth and ammonia-oxidation activity in N. europaea. Furthermore, comparison of viability and activity measurements suggest that the membrane integrity-based stain does not serve as a good indicator of activity. These insights into the responses of pure culture nitrifying bacteria to free chlorine and monochloramine could prove useful in designing disinfection strategies effective in the control of nitrification. / Master of Science

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