The gram-negative endotoxin, lipopolysaccharide (LPS), has been extensively researched in high doses (10-200ng/ml) and is well-documented in the literature for its ability to result in devastating effects such as multi-organ failure, sepsis, and septic shock. In high doses, LPS signals through Toll-like-receptor 4 (TLR4) and triggers a cascade of events culminating in the release of pro- and anti-inflammatory cytokines and the activation of NF-κB. In contrast, super-low doses of LPS (1-100pg/ml) are able to trigger the persistent release of pro-inflammatory mediators while evading the compensatory activation of NF-κB. This mild yet persistent induction of inflammation may lie at the heart of numerous inflammatory diseases and disorders and warrants studies such as this to elucidate the novel mechanisms. In this study, we explored the novel mechanisms utilized by super-low dose LPS in cellular stress and low-grade inflammation. In the first study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and necroptosis were examined. We show that in the presence of super-low dose LPS (50pg/ml), the key regulators of mitochondrial fission and fusion, Drp1 and Mfn1 respectively, are inversely regulated. An increase in mitochondrial fragmentation and cell death which was not dependent on caspase activation was observed. In addition, super-low dose LPS was able to activate RIP3, a kinase responsible for inducing the inflammatory cell death, necroptosis. These mechanisms were regulated in an Interleukin-1 receptor-associated kinase 1 (IRAK-1) dependent manner. In the second study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and endosome/lysosome fusion were examined. In the presence of low-dose LPS (50pg/ml), endosomal-lysosomal fusion is inhibited and a loss of endosomal acidification required for the successful clearance of cellular debris and resolution of inflammation was observed. Additionally, super-low dose LPS induced the accumulation of p62 indicative of the suppression of autophagy. Tollip and Interleukin-1 receptor-associated kinase 3 (IRAK-M) appear to be critical regulators in this process. Collectively, these studies show that low-dose endotoxemia is capable of causing persistent cellular stress, not observed in the presence of high-dose LPS (10-200ng/ml), and that it promotes necroptotic cell death while suppressing mechanisms necessary for the resolution of inflammation such as endosome-lysosome fusion. This research reveals novel mechanisms utilized by low-dose endotoxemia which could aid future efforts to develop prevention and treatment for various debilitating inflammatory diseases. / Ph. D.

Yes / A series of ruthenium and iridium complexes have been synthesized and characterized with 20 novel crystal structures discussed. The library of β-ketoiminato complexes has been shown to be active against MCF-7 (human breast carcinoma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma), and A2780cis (cisplatin-resistant human ovarian carcinoma) cell lines, with selected complexes’ being more than three times as active as cisplatin against the A2780cis cell line. Selected complexes were also tested against the noncancerous ARPE-19 (retinal pigment epithelial cells) cell line, in order to evaluate the complexes selectivity for cancer cells. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is overexpressed in cancer cells, and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anticancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant levels of cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double-strand DNA breaks or DNA cross-linking but induced significant levels of single-strand DNA breaks, indicating a mechanism of action different from that of cisplatin. / Lord RM, Hebden AJ, Pask CM, Henderson IR, Allison SJ, Shepherd SL, Phillips RM, McGowan PC

No description available.

Plant programmed cell death (PCD) is an essential process in plant-pathogen interactions. Importantly, PCD can have contrasting effects on the outcome depending on context. For example, plant PCD in plant-biotroph interactions is clearly beneficial to plants, whereas it could be detrimental to plants in plant-necrotroph interactions. Sclerotinia sclerotiorum is an agriculturally and economically important necrotrophic pathogen. Previous studies have shown that S. sclerotiorum secretes oxalic acid (OA) to enhance Sclerotinia virulence by various mechanisms including induction of PCD in plants. A recent study has also shown that reactive oxygen species (ROS) generation correlates with induction of PCD during disease development. These studies focus on links between ROS, oxalate, and PCD, and how they impact S. sclerotiorum disease development. I examined the involvement of ROS in pathogenic development of S. sclerotiorum. I identified and functionally characterized two predicted S. sclerotiorum NADPH oxidases (Nox1 and Nox2) by RNAi. Both nox genes appear to have roles in sclerotial development, while only Nox1-silenced mutants showed reduced virulence. Interestingly, the reduced virulence of the Nox1-silenced mutant correlated with decreased production of OA in the mutant. This observation suggests that regulation of ROS by S. sclerotiorum Nox1 may be linked to OA. The next study details the phenotype of plants inoculated with an S. sclerotiorum oxalate deficient mutant (A2), which showed restricted growth at the infected site. This response resembles the hypersensitive response (HR), and is associated with plant resistance responses including cell wall strengthening, plant oxidative burst, and induction of defensin genes. Conversely, leaves infected with wild type showed unrestricted spreading of cell death and were not associated with these resistant responses. Furthermore, previous work had shown that a Caenorhabditis elegans anti-apoptotic gene (ced-9) conferred resistance to wild type S. sclerotiorum, while this gene had negligible effects on the phenotype of plant leaves inoculated with A2 mutants. These findings suggest that HR-like cell death by A2 and PCD by wild type S. sclerotiorum may be regulated by different pathways. As a whole, these results reveal the importance of ROS, oxalate, and PCD in Sclerotinia disease development as well as the significance of interplay between them. These studies contribute to the understanding of the underlying mechanisms of Sclerotinia disease.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 52 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 42-49).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 138-170). Also available on the Internet.

Des conditions de carence entrainent une mort cellulaire développementale chez le protiste Dictyostelium discoideum. Dans un système in vitro, des cellules de Dictyostelium sont mises en conditions de carence, puis l'addition des inducteurs DIF-1 ou c-di-GMP conduit à une mort cellulaire vacuolaire. DIF-1 est un polyketide produit par Dictyostelium et induisant la différenciation des cellules pré-tiges. Le dinucléotide cyclique c-di-GMP était connu comme un second messager chez les procaryotes, et comme un déclencheur de l'immunité innée dans des cellules de mammifères. Il a été montré par d'autres que des cellules de Dictyostelium peurent produire et détecter c-di-GMP.Pour analyser la signalisation par c-di-GMP chez Dictyostelium, nous avons utilisé la mutagénèse aléatoire et la mutagénèse ciblée. En utilisant des mutants inactivant stlB ou dmtA, nous avons démontré que DIF-1 endogène ou exogène est nécessaire pour la signalisation par c-di-GMP dans Dictyostelium. En conséquence, nous avons amélioré l'étape de sélection dans une mutagenèse aléatoire en utilisant c-di-GMP et un peu de DIF-1 comme inducteurs, ce qui a produit plusieurs mutants. Par ailleurs j’ai testé par mutagenèse ciblée des hypothèses basées sur les informations connues dans Dictyostelium ou d'autres types de mort cellulaire. Trois molécules ont été essayées, DDX41 comme récepteur putatif de c-di-GMP, l' uniport mitochondrial pour le Ca2+(MCU) et la Na+/K+ATPase (IonA).En résumé, au cours de ma thèse, nous avons démontré une relation entre la signalisation c-di-GMP et a signalisation DIF-1 dans Dictyostelium et identifié plusieurs nouvelles molécules de la mort cellulaire par mutagenèse aléatoire. / The protist Dictyostelium discoideum undergoes development cell death when under starvation. To investigate the molecular mechanism of Dictyostelium cell death, an in vitro system has been used. Dictyostelium cells were starved and then cell death was induced by DIF-1 or c-di-GMP. About 40h after induction, cells underwent vacuolar cell death. DIF-1 is a polyketide, produced by Dictyostelium prespore cells, which induces prestalk cell differentiation. c-di-GMP was well known not only as a second messenger produced and sensed by bacteria but also as a trigger of innate immunity in mammalian cells. Dictyostelium was recently found by another laboratory to produce and sense c-di-GMP. To analyze c-di-GMP signaling in Dictyostelium cell death, we used random mutagenesis and targeted mutagenesis. By using the knockout mutants stlB- and dmtA-, we demonstrated that endogenous or exogenous DIF-1 is required for c-di-GMP signaling in Dictyostelium. In contrast, endogenous c-di-GMP is not necessary for exogenous DIF-1 signaling. As a consequence, we improved the selection step in random mutagenesis by using c-di-GMP and a little DIF-1 as inducers, which produced several mutants. Another part of my project was to test by targeted mutagenesis some hypotheses, based on known information in Dictyostelium or other similar cell death types. Three molecules have been tested, the c-di-GMP putative receptor DDX41, the mitochondrial Ca2+ uniporter (MCU) and the Na+/K+-ATPase (IonA).In summary, during my thesis, we have demonstrated a relation between c-di-GMP signaling and DIF-1 signaling in Dictyostelium and identified several new cell death molecules by random mutagenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21303号 / 医博第4392号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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