Cell death is a fundamental aspect of development and homeostasis; its dysregulation is commonly associated with disease. Historically, apoptosis has been the most heavily studied type of cell death, but there are many other non-apoptotic forms of cell death. The Drosophila ovary provides a powerful in vivo model to study non-apoptotic cell death. Each egg chamber in the ovary contains 15 nurse cells that support an oocyte throughout development, and at the end of oogenesis the nurse cells are surrounded by stretch follicle cells and undergo non-apoptotic cell death. The work in this dissertation investigated the role of stretch follicle cells in nurse cell death. Genetic ablation of the stretch follicle cells revealed that they are required for multiple nurse cell death events including the transport of cytoplasm to the oocyte and DNA fragmentation. We found that phagocytic machinery is required in the stretch follicle cells for the acidification and elimination of nurse cells, suggesting nurse cells die by phagoptosis. Furthermore, live imaging and a transgenic engulfment detector demonstrated that nurse cells are not engulfed piece-wise despite the requirement of phagocytosis machinery, but are instead surrounded and acidified extracellularly. To determine the mechanism driving nurse cell acidification, we performed a targeted RNAi screen against lysosome-associated genes. Using tissue-specific RNAi, we demonstrated that the V-ATPase proton pump is required in the stretch follicle cells for nurse cell acidification. GFP fusion proteins and antibody staining revealed that V-ATPases become enriched and localize to the stretch follicle cell plasma membranes to acidify the nurse cells that they surround. Following acidification, the stretch follicle cells were found to release cathepsins, lysosomal proteases, to break down and degrade the nurse cell. To uncover novel pro-death proteins that mediate signaling between the stretch follicle cells and nurse cells, we utilized proximity-dependent protein labeling and identified proteins enriched in the stretch follicle cells. Altogether this work uncovers a new role for lysosomal machinery acting at the plasma membrane of stretch follicle cells to drive nurse cell death, and identifies pro-death proteins in the stretch follicle cells that promote nurse cell death.

No description available.

No description available.

Tumour-associated macrophages (TAMs) have been shown to play an important role in tumour survival and progression. Thus, high numbers of macrophages in the tumour tissue are often associated with a poor prognosis. Identification of factors responsible for recruiting macrophages to the sites of different types of tumours might help to develop more effective cancer treatment. Burkitt's lymphoma (BL) is characterised by uncontrolled cell proliferation, high rate of spontaneous apoptosis and significant macrophage infiltration. Although BL cells undergo extensive apoptosis, in situ their corpses are cleared very effectively by macrophages infiltrating the tumour. It is now widely believed that dying cells are themselves able to release chemotactic molecules to ensure macrophage chemotaxis and subsequent clearance of their site of death. Previous work carried out in this laboratory identified fractalkine/CX3CL1 (FKN) released from dying BL cells to be an important player in macrophage chemotaxis to BL. Yet, these results have also indicated that FKN may not be the only chemokine involved in this process. Following from those observations, the first part of this work focused on examination of the potential role of monocyte chemoattractant protein-1 (MCP-1) in macrophage recruitment to BL. Despite the initial promising results, careful analysis of the data obtained by various techniques led to the conclusion that MCP-1 is, probably, not expressed by BL cells. Subsequently, effort was concentrated on understanding mechanisms regulating FKN processing during cell death. The studies performed before in this laboratory identified a new form of FKN to be present in apoptotic BL cells and showed that this is the form that is, most likely, responsible for mediating macrophage migration. Here, this apoptosis-related 60 kDa FKN was found to be a likely caspase-3 cleavage product. Moreover, it was demonstrated that FKN and active caspase-3 are released together in apoptotic BL cell-derived microparticles, suggesting that the proteolytic events could take place also extracellularly. In the final results chapter the differences between BL cell lines in the way they process FKN during cell death were revealed and a new cell death-associated 55 kDa FKN was observed. Through several lines of evidence, this new form was identified to be a possible product of calpain-mediated proteolysis. To conclude, this work provides the first evidence for a possible direct participation of the two major cell death executioner proteases – caspases and calpains, in production of ‘find me’ signals for macrophages and thus, ensuring effective clearance of dying cells. These results indicate that FKN cleavage and release might be of key importance during cell death. Moreover, the studies presented here contribute to better understanding of the process of FKN secretion.

This thesis comprises two parts, both concerned with the study of Escherichia coli bacterial suspensions. The first part investigates E. coli motility whilst the second part explores E. coli growth in the presence of the antimicrobial peptide pexiganan. In Part 1 I measure the three-dimensional diffusion of non-motile cells in an active suspension of E. coli, using Differential Dynamic Microscopy (DDM). It is found that tracer diffusivity is enhanced linearly as a function of the bath activity, defined as the product of the number density of active bacteria and their average speed. The absolute enhancement is measured to be 1:8 ± 0:1 times smaller that that published previously in the vicinity of a surface, in agreement with theoretical predictions of enhanced diffusion by far-field advection. The diffusivities of non-motile mutants with and without paralysed flagella are enhanced to the same extent, despite a difference in hydrodynamic radii. In addition, the protocol for growing, preparing and measuring motile E. coli is optimised using DDM. In Part 2 I investigate how E. coli density in liquid media supplemented with pexiganan influences the measurement of its Minimum Inhibitory Concentration (MIC). Growth curves, peptide bioassays and single cell microscopy are used. It is found that population density drops rapidly when pexiganan is introduced, but regrowth occurs within 24 hours at sub-MIC concentrations. The shape of the density curve is explained by peptide depletion linked to cell death and immediate recovery of cells exposed to the peptide. As expected from these findings, the system displays a substantial inoculum effect, quantified with a fitted power law. Substantial variation is seen between replicate MIC assays; an inherent property of the system which derives from the drop to small numbers of viable cells before regrowth. Finally, I show that DDM measurements of E. coli motility in antimicrobial peptides can provide an alternative, high-throughput density curve.

Cell death is a key component in development and for the continued renewal of tissues. Phagoptosis is a process in which phagocytes directly lead to the death of other cells. This process of cell death is significantly less characterized when compared to other mechanisms of cell death, such as apoptosis. In the Drosophila ovary, phagoptosis appears to play a key role in the developmental process of oogenesis. Recent studies have shown that genes associated with phagocytosis are required for the programmed death of nurse cells in the Drosophila ovary. Ovaries are made up of 15 nurse cells, a single oocyte, and a layer of follicle cells bordering them. During the process of egg chamber development, all of the nurse cells undergo programmed cell death. During late oogenesis, each nurse cell is surrounded by a group of follicle cells referred to as stretch follicle cells. These stretch follicle cells have recently been implicated as a main promoter of nurse cell phagoptosis. However, an exact mechanism to explain how these stretch follicle cells induce nurse cell death is not fully characterized. To achieve a more detailed understanding of this mechanism, we are examining the function of the cytoskeleton in this process via live imaging. We hypothesize that the follicle cell cytoskeleton plays a significant role in nurse death due to the importance of actin during phagocytosis. Further, we intend to use these live imaging studies to investigate the role of calcium before, during, and after clearance of the nurse cells. Previous studies have shown that calcium bursts within the cell are associated with the initiation of phagocytosis in macrophages, as well as other phagocytic cell types. Studies in this thesis were done by utilizing live imaging and have shown dynamic changes in follicle cell actin before and during the death of nurse cells. These confocal microscopy real time videos have revealed that follicle cell actin polymerizes towards the nurse cell immediately before acidification. Following acidification of the nurse cells, the follicle cell actin changes direction, moving towards the phagocytic follicle cell. Additionally, through live imaging we have observed calcium bursts in the follicle cells immediately before nurse cell death. Overall, this work has provided a more detailed understanding of nurse cell death.

Orientador: João Martins Pizauro Júnior / Banca: Marcia Regina Cominetti / Banca: Marcos Túlio de Oliveira / Banca: Andrei Leitão / Banca: Luis Henrique Souza Guimarães / Resumo: A metamorfose dos anfíbios é um processo altamente regulado que envolve a participação de hormônios e biomoléculas na morte celular e absorção da cauda, a qual libera nutrientes para as transformações morfofisiológicas necessárias para a ocupação do meio terrestre. Dentre os nutrientes liberados, o fosfato é essencial para a síntese de ATP, membranas biológicas, ácidos nucleicos e metabólitos fosforilados, bem como para a regulação de processos degradativos e da atividade de enzimas. Neste sentido, a identificação e caracterização cinética das fosfatases que participam da absorção da cauda de girinos de Lithobates catesbeianus, durante a passagem da fase larval aquática para a adulta terrestre, poderão contribuir para a compreensão dos eventos envolvidos na morte celular e na liberação de nutrientes. O estudo revelou aumento da atividade das fosfatases ácida e alcalina, de aproximadamente 30 e 17 vezes, respectivamente, no período da metamorfose. A centrifugação diferencial do extrato bruto da cauda e os estudos cinéticos, permitiram a identificação de três fosfatases distintas, sendo uma fosfatase ácida solúvel, uma fosfatase ácida ligada à membrana e uma fosfatase alcalina ligada à membrana por meio da âncora de fosfatidilinositol. A fosfatase ácida ligada à membrana revelou Vm=104,5 U.mg-1; Km=0,29 mM e nH=0,9; a solúvel, Vm= 25,9 U.mg-1; Km=0,33 mM e nH =0,8, enquanto a fosfatase alcalina ligada à membrana revelou Vm=10,5 U.mg-1; Km=0,2 mM e nH=1,0, para a hidrólise do pN... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Amphibian metamorphosis is a tightly regulated transformation that involves the participation of hormones and other biomolecules in cell death and tail absorption, to release nutrients for the morphophysiological changes necessary for the occupation of the terrestrial environment. Among these nutrients, the phosphate is essential for the synthesis of ATP, biological membranes, nucleic acids and phosphorylated metabolites; regulation of degradative processes and of enzymes activity. In this sense, the identification and characterization of the phosphatases that participate in the absorption of the tail of Lithobates catesbeianus during the transition from the aquatic larval phase to the terrestrial adult, may contribute to the understanding of the events involved in cell death and nutrient release.The study showed that there was an increase in the activity of the acid and alkaline phosphatases of approximately 30 and 17 fold, respectively, in the metamorphosis period. The differential centrifugation of the tail crude extract, as well as the kinetic tests, allowed the identification of three distinct phosphatases, a soluble acid phosphatase, a membrane bound acid phosphatase and an alkaline phosphatase bound to the membrane by the anchor of phosphatidylinositol. The kinetic characterization of membrane bound acid phosphatase revealed Vm=104.5 U.mg-1; K0.5=0.29 mM and nH=0.9; the soluble, Vm= 25.9 U.mg-1; K0.5=0.33 mM and nH =0.8, while the membrane bound alkaline phosphatase revealed Vm=10.5 U.mg-1; K0.5=0.2 mM and nH=1.0, for pNPP hydrolysis. The mass spectrometry results showed the presence of three protein phosphatases, two serine/threonine, being one PP2A, one PP1 and one tyrosine, PTP-LMW. Studies using specific substrates for PP2A and PTP-LMW and inhibitor for PP1 confirmed their presence in the tail of the tadpoles of L. catesbeianus. In addition, the separation in different protein fractions, allowed... / Doutor

L’efficacité de certains agents anti-cancéreux, notamment les anthracyclines et l’oxaliplatine repose sur l'induction d’une mort cellulaire immunogène (MCI) pouvant conduire à une réponse immunitaire anti-tumorale spécifique. Les cellules succombant à ce type particulier d'apoptose vont subir certaines modifications définies par un modèle spatio-temporel précis. Celui-ci est caractérisé par la mise en place de signaux d’apparition séquentielle, dont le plus précoce est l’exposition membranaire d’une protéine du réticulum endoplasmique, la calréticuline (CRT) qui constitue un signal de danger essentiel à la phagocytose des cellules mourantes par les cellules dendritiques. Ensuite, à un stade apoptotique, la sécrétion d’adénosine triphosphate (ATP) dépendante de l'autophagie active l’inflammasome NLRP3 et induit la polarisation des cellules T CD8+ productrices d’IFN-. Enfin, au cours de la nécrose secondaire, le relargage d’un facteur pro-immunogène High-mobility group protein B1 (HMGB1) est indispensable à une présentation antigénique optimale aux cellules T CD4+ et CD8+, contribuant ainsi à l’activité tumoricide de la chimiothérapie et protégeant l’hôte d’une éventuelle rechute. De manière à identifier de nouvelles molécules capables d’induire une réponse immunitaire anti-tumorale spécifique, un criblage à haut débit de bibliothèques de composés approuvés par la FDA (Food and Drug Administration) a été réalisé grâce à l’utilisation de microscopie automatisée et de biosenseurs permettant la détection de l'exposition de la CRT, de la sécrétion d'ATP et du relargage d'HMGB1. Ce criblage multiparamétrique à haut débit a permis d’identifier les glucosides cardiotoniques (GCs), déjà bien connus pour leur activité cytotoxique préférentielle des cellules cancéreuses, comme étant des inducteurs efficaces de la MCI. Cette découverte a été validée par des méthodes alternatives in vitro, suivis d’une étude de la mécanistique d’induction de la MCI par les GCs. Les résultats ont mis en évidence une inhibition spécifique de la sous-unité α1 de la pompe Na + / K + ATPase, qui à son tour modifie l'homéostasie calcique de la cellule cible, un effet reproduit par les ionophores du Ca2 +. Nous avons ensuite montré que les CGs, en combinaison avec des chimiothérapies non immunogènes (cisplatine ou mitomycine C) pouvaient vacciner des souris syngéniques contre une ré-injection de cellules cancéreuses vivantes et que les effets antinéoplastiques de ces agents endommageants l’ADN pouvaient être potentialisés par les GCs dans les hôtes immunocompétents mais pas dans les souris immunodéficientes. Enfin, une analyse rétrospective de patients atteints de carcinomes et traités par un GC couramment utilisé en clinique dans la prise en charge de l'insuffisance cardiaque, la digoxine (n=145) a révélé une amélioration significative de la survie globale par rapport à celle de patients non traités (n=290). Les patients ont été appariés en fonction de leur âge, sexe, type de cancer et principaux paramètres pronostiques. Des analyses plus approfondies ont ensuite révélées que la digoxine n’affectait pas la survie globale des patients déjà traités par des agents chimiothérapeutiques immunogènes mais celle des patients ayant reçu des agents autres que les anthracyclines ou l’oxaliplatine. / The efficacy of some anti-cancer agents, including anthracyclines and oxaliplatin is based on their capacity to induce immunogenic cell death (ICD) in tumor cells. This peculiar type of apoptosis is defined by a sequential emission of specific immunogenic signals from the dying tumor, which in their correct spatio-temporal appearance ignite a specific immune response against therapy resistant and dormant tumor (stem) cells. Thus the early membrane exposure of the ER-resident molecular chaperone calreticulin (CRT) constitutes a critical uptake signal for the engulfment of dying tumor cells by dendritic cells (DCs). Then, at later stages, the autophagy-dependent secretion of ATP and its binding to purinergic receptors on DCs activates the NLRP3 inflammasome. Subsequent release of IL-1 by DC triggers the polarization of IFN-γ producing CD8+ T cells. Finally, during secondary necrosis, the release of the pro-immunogenic high-mobility group box 1 (HMGB1) protein and its interaction with Toll like receptor 4 (TLR4) on DCs facilitates an optimal antigen presentation to T cells. Thus ICD contributes to the tumoricidal activity of chemotherapy and protecting the host from relapse. In order to identify thus far unknown inducers of ICD, a high content screening of compound libraries approved by the Food and Drug Administration (FDA) was conducted by means of robotized automated bioimaging combined with ICD biosensors allowing for the detection of CRT relocation, ATP secretion and HMGB1 release. This multiparametric approach led to the identification of cardiac glycosides (CGs), already well known for their preferential cytotoxic activity on cancer cells, as effective inducers of ICD. The hit compounds were validated by alternative methods in vitro, followed by a mechanistic study of GC induced ICD. Results indicated an on-target inhibition of the Na+/K+ ATPase subunit 1, which in turn interfered with the Ca2+-homeostasis of the target cell, an effect that could be mimicked by Ca2+ ionophores. We then showed in different mouse models that tumor cells killed with a combination of GC and non-immunogenic chemotherapy (cisplatin or mitomycin C) have the ability to immunize syngeneic mice against rechallenge with living cells. In addition the antineoplastic effects of these DNA damaging agents in vivo were increased by GCs in immunocompetent but not in immunodeficient mice. Finally, retrospective clinical analyses revealed that the administration of the GC digoxin during chemotherapy had a significant positive impact on overall survival in cohorts of breast, colorectal, head and neck, and hepatocellular carcinoma patients, especially when they were treated with agents other than anthracyclines and oxaliplatin.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. Previous studies have demonstrated that the sexually transmitted pathogen, Neisseria gonorrhoeae, can induce or inhibit host cell death. N. gonorrhoeae is a mucosal pathogen and, in females, initiates infection in epithelial cells of the ectocervix and endocervix. Mucosal epithelial cells of the female genital tract are the first line of defense, and thus their cellular fate can alter the early immune response to invading pathogens such as N. gonorrhoeae. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In the present study, we demonstrate that N. gonorrhoeae stimulation induces a transient increase in cIAP2 protein levels in human cervical epithelial cells. High intracellular protein levels were observed during early N. gonorrhoeae stimulation and were followed by a marked intracellular decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes, which are nano-sized vesicles that carry protein and coding RNA as cargo from one cell to another. We also observed that depletion of cIAP2 in N. gonorrhoeae stimulated cells resulted in cell death resembling necroptosis, an inflammatory form of cell death. Furthermore, inhibition of cIAP2 led to an increase in interleukin-1β production. Exosomes have been found to have important roles in cell communication during microbial infection. Here, we demonstrate that N. gonorrhoeae induces exosome production and alters exosome content. We also demonstrate that exosomes elicit cytokine responses in uninfected naïve cells. Collectively, these studies highlight an additional mechanism for epithelial cells to orchestrate the immune response in the female genital tract during N. gonorrhoeae infection.

by Kwan, Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 108-116). / Abstract --- p.i / Achnowledgment --- p.ii / List of abbreviations --- p.iii / List of table and figures --- p.v / Table of contents --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- THE DISCOVERY OF TUMOUR NECROSIS FACTOR (TNF) --- p.1 / Chapter 1.2 --- THE MOLECULE AND ITS RECEPTORS --- p.1 / Chapter 1.3 --- THE BIOLOGICAL ACTIVITIES OF TNF --- p.3 / Chapter 1.4 --- STUDIES ON THE CYTOTOXIC MECHANISM OF TNF --- p.4 / Chapter 1.5 --- A TENTATIVE MECHANISM OF TNF CYTOTOXICITY --- p.11 / Chapter 1.6 --- THE GLUTATHIONE SYSTEM : A CELLULAR PROTECTIVE MECHANISM AGAINST OXIDATIVE STRESS …… --- p.12 / Chapter 1.7 --- OBJECTIVE AND STRATEGY OF THIS STUDY --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND APPARATI / Chapter 2.1 --- CELL LINES --- p.19 / Chapter 2.2 --- "ISOLATION, MAINTENANCE AND SUBCULTURE OF CELL LINES" --- p.19 / Chapter 1. --- Plain RPMI-1640 medium / Chapter 2. --- Penicillin-streptomycin solution / Chapter 3. --- Foetal bovine serum / Chapter 4. --- Complete RPMI-1640 medium / Chapter 5. --- Trypsin-ethylenediaminetetraacetate solution / Chapter 6. --- Phosphate buffered saline / Chapter 7. --- Cycloheximide / Chapter 8. --- Actinomycin D / Chapter 9. --- Trypan blue stain / Chapter 10. --- Neutral red stain / Chapter 11. --- Recombinant human tumour necrosis factor / Chapter 12. --- Cell culture plates and flasks / Chapter 2.3 --- GROWTH CHARACTERISTIC --- p.22 / Chapter 1. --- Tritiated Thymidine / Chapter 2. --- Tritiated Leucine / Chapter 3. --- Trichloroacetic acid / Chapter 4. --- Scintillation cocktail / Chapter 2.4 --- "RESPONSE TOWARDS ANTICANCER DRUGS, CYTOTOXIC AGENTS, AND ENZYME MODULATORS" --- p.23 / Chapter 1. --- N-acetyl-DL-homocysteinethiolactone / Chapter 2. --- Diethyldithiocarbamic acid / Chapter 3. --- Doxorubicin / Chapter 4. --- Acivicin / Chapter 5. --- Ethacrynic acid / Chapter 6. --- "L'Buthionine-[S,R]-sulfoximine" / Chapter 7. --- Hydrogen peroxide / Chapter 8. --- Methotrexate / Chapter 9. --- Menadione / Chapter 2.5 --- CULTURE OF BACTERIAL CELLS --- p.27 / Chapter 1. --- Ampicillin stock solution / Chapter 2. --- Chloramphenicol stock solution / Chapter 3. --- Tetracycline stock solution / Chapter 4. --- Luria-Bertani medium / Chapter 5. --- LB with ampicillin / Chapter 6. --- SOB medium / Chapter 7. --- SOB medium with ampicillin / Chapter 8. --- SOC medium / Chapter 9. --- SB medium / Chapter 10. --- SB medium with ampicillin / Chapter 11. --- Agar plates / Chapter 2.6 --- PREPARATION OF DNA PROBES FROM BACTERIAL CLONES --- p.29 / Chapter 1. --- FlexiPrep Kit / Chapter 2. --- Restriction endonucleases / Chapter 3. --- GeneClean® II Kit / Chapter 4. --- cDNA clones for making DNA probes / Chapter 5. --- TrisHCl EDTA buffer / Chapter 2.7. --- ELECTROPHORESIS OF DNA --- p.31 / Chapter 1. --- EDTA stock solution / Chapter 2. --- Tris acetate EDTA electrophoresis buffer / Chapter 3. --- Tris borate EDTA electrophoresis buffer / Chapter 4. --- Ethidium bromide / Chapter 5. --- DNA molecular size markers / Chapter 6. --- TAE/TBE agarose gel slab / Chapter 2.8 --- CONSTRUCTION OF MURINE TNFR1 PARTIAL cDNA CLONE --- p.33 / Chapter 1. --- Frist strand cDNA synthesis Kit / Chapter 2. --- Murine TNFR1 forward and reverse primers / Chapter 3. --- Polymerase chain reaction reagents / Chapter 4. --- Cloning vector / Chapter 5. --- Modifing enzymes / Chapter 6. --- T7 SequencingTM Kit / Chapter 7. --- Acrylamide/bis gel stock solution / Chapter 8. --- Urea / Chapter 9. --- TEMED and ammonium persulphate / Chapter 10. --- β-Galactosidase colour test reagents / Chapter 11. --- TFB solution / Chapter 12. --- DnD solution / Chapter 2.9. --- RADIOLABELLING OF DNA PROBES --- p.35 / Chapter 1. --- Oligolabelling kit / Chapter 2. --- Redivue [α-32P] dCTP / Chapter 3. --- PUSH column / Chapter 2.10 --- EXTRACTION OF TOTAL RNA FROM CELL LINES --- p.36 / Chapter 1. --- N-Lauroylsarcosine / Chapter 2. --- 2M sodium acetate (pH48) / Chapter 3. --- Phenol / Chapter 4. --- Isopropanol / Chapter 5. --- Ethanol / Chapter 6. --- Extraction buffer / Chapter 7. --- Chloroform / Chapter 8. --- Isoamyl alcohol / Chapter 2.11 --- HYBRIDIZATION AND NORTHERN ANALYSIS --- p.37 / Chapter 1. --- 20XSSC / Chapter 2. --- 5X formaldehyde running buffer / Chapter 3. --- RNA sample buffer / Chapter 4. --- 10X RNA loading buffer / Chapter 5. --- Formaldehyde slab gel / Chapter 6. --- Hybond®-N membrane / Chapter 7. --- Immobilon®-N membrane / Chapter 8. --- Salmon sperm DNA / Chapter 9. --- Sodium dodecyl sulphate / Chapter 10. --- Dextran sulphate / Chapter 11. --- Kodak Biomax MR and X-OMAT films and developing kits / Chapter 2.12 --- APPARATI USED --- p.39 / Chapter CHAPTER 3 --- METHODS / Chapter 3.1 --- ISOLATION AND MAINTENANCE OF TNF RESISTANT L929 CELLS --- p.40 / Chapter 3.1.1 --- Culture of L929 cells / Chapter 3.1.2 --- Trypan blue exclusion test / Chapter 3.1.3 --- Isolation of TNF-resistant variants of L929 / Chapter 3.1.4 --- Verification of the TNF-resistant trait of rL929 / Chapter 3.1.5 --- Neutral red uptake assay / Chapter 3.2 --- COMPARING L929 AND rL929 CELLS IN TERMS OF GROWTH CHARACTERISTICS --- p.43 / Chapter 3.2.1 --- Doubling time / Chapter 3.2.2 --- Rate of protein synthesis / Chapter 3.2.3 --- Rate of DNA synthesis / Chapter 3.3 --- COMPARING L929 AND rL929 CELLS IN TERMS OF RESPONSE TOWARDS DIFFERENT ENZYME INHIBITORS AND CYTOTOXIC AGENTS --- p.44 / Chapter 3.3.1 --- TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.2 --- Effects of inhibitors of gene transcription and protein synthesis on TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.3 --- Cytotoxic effect of hydrogen peroxide and menadione on L929 and rL929 cells --- p.44 / Chapter 3.3.4 --- TNF cytotoxicity on L929 and rL929 cells: effect of N-acetyl homocysteine thiolatone --- p.45 / Chapter 3.3.4.1 --- The tolerant limit of AHCT / Chapter 3.3.4.2 --- Effect of AHCT on TNF cytotoxicity / Chapter 3.3.5 --- TNF cytotoxicity on L929 and rL929 cells: effect of diethyldithiocarbamate --- p.46 / Chapter 3.3.5.1 --- The tolerant limit of DEDTC / Chapter 3.3.5.2 --- Effect of DEDTC on TNF cytotoxicity / Chapter 3.3.6 --- TNF cytotoxicity on L929 and rL929 cells: effect of buthionice sulfoximine --- p.47 / Chapter 3.3.6.1 --- The tolerant limit of BSO / Chapter 3.3.6.2 --- Effect of BSO on TNF cytotoxicity / Chapter 3.3.7 --- TNF cytotoxicity on L929 and rL929 cells: effect of Acivicin --- p.47 / Chapter 3.3.7.1 --- The tolerant limit of acivicin / Chapter 3.3.7.2 --- Effect of acivicin on TNF cytotoxicity / Chapter 3.3.8 --- TNF cytotoxicity on L929 and rL929 cells: effect of ethacrynic acid --- p.48 / Chapter 3.3.8.1 --- The tolerant limit of ethacrynic acid / Chapter 3.3.8.2 --- Effect of ethacrynic acid on TNF cytotoxicity / Chapter 3.3.9 --- Cytotoxic effect of doxorubicin on L929 and rL929 cells --- p.49 / Chapter 3.3.10 --- TNF cytotoxicity of L929 cells: effect of N-acetyl cysteine --- p.49 / Chapter 3.3.11 --- Cytotoxic effect of methotrexate on L929 and rL929 cells --- p.50 / Chapter 3.3.12 --- Cytotoxic effect of hyperthermia on L929 and rL929 cells --- p.50 / Chapter 3.4 --- NORTHERN ANALYSIS AND HYBRIDIZATION --- p.51 / Chapter 3.4.1. --- Preparing RNA blots --- p.51 / Chapter 3.4.1.1 --- Extraction of total RNA from cells / Chapter 3.4.1.2 --- Making equal loading of RNA samples in formaldehyde gel electrophoresis / Chapter 3.4.1.3 --- Northern blotting of RNA / Chapter 3.4.2. --- Preparation of cDNA probes --- p.53 / Chapter 3.4.2.1 --- Preparing plasmids from A TCC clones / Chapter 3.4.2.2 --- Preparing TNFR1 probe from first-strand cDNA of L929 cells / Chapter 1. --- Construction of recombinant clone from the PCR product of TNFRl fragment / Chapter 2. --- Transforming the recombinant vector into JM109 host / Chapter 3. --- Sequencing of PCR product for identity confirmation / Chapter 3.4.2.3 --- Preparing DNA inserts from plasmids / Chapter 3.4.3 --- Radiolabelling of cDNA probes --- p.56 / Chapter 3.4.4 --- Hybridization of radioactive probes to RNA blots --- p.57 / Chapter CHAPTER 4 --- RESULTS AND DISCUSSIONS / Chapter 4.1 --- ISOLATION OF TNF-RESISTANT VARIANTS OF L929 CELLS --- p.58 / Chapter 4.1.1 --- Single cell subcloning of TNF-resistant L929 variants / Chapter 4.1.2 --- Growth rates of L929 and rL929 cells / Chapter 4.1.3 --- Rate of protein synthesis in L929 and rL929 cells / Chapter 4.1.4 --- Rate of DNA synthesis in L929and rL929 cells / Chapter 4.2 --- EFFECT OF INHIBITORS OF GENE TRANSCRIPTION AND PROTEIN SYNTHESIS ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.67 / Chapter 4.3 --- RESPONSE OF L929 AND rL929 CELLS TOWARDS VARIOUS CYTOTOXIC AGENTS --- p.70 / Chapter 4.3.1 --- "Response towards methotrexate, an anti-metabolite used in cancer treatment" / Chapter 4.3.2 --- "Response towards doxorubicin, an chemotherapeutic agent used in cancer treatment" / Chapter 4.3.3 --- "Response towards menadione, a cytotoxic agent known to generate free radicals inside cells" / Chapter 4.3.4 --- Response towards hydrogen peroxide: a highly oxidative agent / Chapter 4.3.5 --- "Response towards hyperthermia, a treatment known to exert oxidative stress on cells" / Chapter 4.4 --- EFFECTS OF MODULATORS OF CYTOSOLIC SUPEROXIDE DISMUTASE ON TNF CYTOTOXICITY ON L929 and rL929 CELLS --- p.77 / Chapter 4.5 --- EFFECT OF MODULATORS OF GLUTATHIONE METABOLISM ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.82 / Chapter 4.5.1 --- "Effects of L-buthionine [S,R] sulfoximine, an inhibitor of glutathione synthesis" --- p.82 / Chapter 4.5.2 --- "Effect of N-acetyl cysteine, a cysteine derivative" --- p.84 / Chapter 4.5.3 --- "Effects of acivicin , an inhibitor of GSH reuptake and recycle" --- p.85 / Chapter 4.5.4 --- "Effect of ethacrynic acid, an inhibitor of glutathione S- transferase" --- p.87 / Chapter 4.6 --- GENE EXPRESSION IN L929 AND rL929 CELLS IN THE COURSE OF TNF CHALLENGE --- p.89 / Chapter 4.6.1 --- Isolation of total RNA from L929 and rL929 cells --- p.89 / Chapter 4.6.2 --- Preparation of DNA probes for hybridization --- p.89 / Chapter 4.6.3 --- Hybridization of specific probes on RNA blots --- p.90 / Chapter 4.6.3.1 --- Expression of heat shock protein --- p.70 / Chapter 4.6.3.2 --- Expression of the p55 TNF receptor / Chapter 4.6.3.3 --- Expression of glutathione reductase / Chapter 4.6.3.4 --- Expression of glutathione S-transferase pi / Chapter 4.7 --- DISCUSSIONS OF THE EXPERIMENTAL RESULTS --- p.97 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.104 / APPENDIX / Generation of the TNF receptor 1 cDNA probe --- p.106 / REFERENCES --- p.108