by Chan Chun-wai, Francis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 105-116). / Abstract --- p.I / Acknowledgments --- p.III / Abbreviations --- p.IV / Objectives --- p.VI / Content --- p.VII / Chapter Section 1 --- Introduction / Chapter I. --- Preamble --- p.1 / Chapter II. --- Characteristics of Cell Death Process --- p.1 / Chapter II.1. --- Necrosis --- p.1 / Chapter II.2. --- Apoptosis-Programmed Cell Death --- p.5 / Chapter III. --- Triggering of Programmed Cell Death --- p.10 / Chapter IV. --- DNA Fragmentation and Activation of Endogenous Endonuclease --- p.12 / Chapter V. --- Signal Transduction Leading to Programmed Cell Death --- p.14 / Chapter V.1. --- Role of Calcium Ion --- p.14 / Chapter V.2. --- Role of Protein Kinase C --- p.15 / Chapter V.3. --- Protein Dephosphorylation by Phosphatases --- p.16 / Chapter V.4. --- Role of Adenosine 3':5'-cyclic Monophosphate --- p.17 / Chapter V.5. --- Other Signaling Mechanisms --- p.17 / Chapter VI. --- Gene Regulation in Programmed Cell Death --- p.19 / Chapter VI. 1. --- Gene Expression in Programmed cell death --- p.19 / Chapter VI. 1.1 . --- Tissue Transglutaminase --- p.19 / Chapter VI. 1.2. --- Poly (ADP-ribose) Polymerase --- p.20 / Chapter VI. 1.3. --- Testosterone-Repressed Prostate Message-2 Gene --- p.20 / Chapter VI. 1.4. --- Other Programmed Cell Death Associated Gene Expressions --- p.21 / Chapter VI.2. --- Protooncogene Regulation in Programmed Cell Death --- p.22 / Chapter VI.2.1. --- bcl-2 Expression --- p.22 / Chapter VI.2.2. --- c-myc Expression --- p.23 / Chapter VII. --- Concanavalin A and succinylated Concanavalin A --- p.25 / Chapter VII. 1. --- Physiochemical Characterization --- p.25 / Chapter VII.2. --- Cellular Response to Concanavalin A --- p.29 / Chapter VIII. --- Features of Murine Macrophage Cell Line PU5-1.8 and Normal Macrophages --- p.32 / Chapter Section 2 --- Materials and Methods / Chapter I. --- Materials --- p.33 / Chapter II. --- Cell Culture --- p.33 / Chapter III. --- [Methyl-3H]-Thymidine Incorporation Assay --- p.34 / Chapter IV. --- [Methyl-3H]-Thymidine Release Assay --- p.34 / Chapter V. --- "3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ) Cell Death Assay" --- p.35 / Chapter VI. --- Identification of Cell Death using DNA Chelating Fluorescence Probes´ؤFluorescent Microscopy and Confocal Laser Microscopy --- p.35 / Chapter VII. --- Analysis of DNA Fragmentation --- p.37 / Chapter VIII. --- Determination of Fluxes by Confocal Laser Microscopy --- p.38 / Chapter IX. --- Determination of PKC Activation by Western Blotting and Immunocytochemistry --- p.39 / Chapter X. --- Statistical Analysis --- p.41 / Chapter Section 3 --- Results / Chapter I. --- Concanavalin A was a Cell Death Causing Agent in PU5-1.8 cells --- p.42 / Chapter I.1 --- Con A Reduced the Cell Proliferation in PU5-1.8 cells --- p.42 / Chapter I.2. --- Con A Exhibited Cytotoxic Effect to PU5-1.8 cells --- p.44 / Chapter I.3. --- Con A Exhibited Cytotoxic Effect on Normal Peritoneal Macrophages --- p.46 / Chapter I.4. --- Succinylated Concanavalin A Showed a Weaker Cytotoxic Effect in the PU5-1.8 cells --- p.46 / Chapter I.5. --- α-D-Methylmannopyranoside Inhibited the Cytotoxic Effect of Con A in PU5-1.8 cells --- p.50 / Chapter I.6. --- FCS Inhibited the Con A-induced cell death of PU5-1.8 cells --- p.52 / Chapter II. --- Concanavalin A was an Apoptosis Causing Agentin PU5-1.8 cells --- p.57 / Chapter II. 1. --- Con A Induced Apoptosis in PU5-1.8 cells --- p.57 / Chapter II. 2. --- Con A Enhanced the Release of DNA in PU5-1.8 cell --- p.63 / Chapter II. 3. --- Con A Induced DNA fragmentation in PU5-1.8 cells --- p.63 / Chapter II.4. --- Cycloheximide Inhibited the Con A-Induced Cell Death in PU5-1.8 cells --- p.67 / Chapter II.5. --- Nicotinamide Inhibited the Con A-Induced Cell Death in PU5-1.8 cells --- p.71 / Chapter III. --- Signaling elicited by Concanavalin A --- p.74 / Chapter III.1. --- Con A Increased Intracellular Free Calcium Ion Concentration of PU5-1.8 cells --- p.74 / Chapter III. 1.1. --- Con A Induced Ca2+ Mobilization in PU5-1.8 cells --- p.74 / Chapter III. 1.2. --- Con A Induced the Ca2+ Influx and Intracellular Ca2+ Mobilization --- p.78 / Chapter III. 1.3. --- BAPTA-AM Inhibited the Ca2+ Mobilization in PU5-1.8 cells Stimulated by Con A --- p.80 / Chapter III.2. --- Role of Protein kinase C --- p.86 / Chapter III.2.1. --- Con A Increased the amount of PKC in PU5-1.8 cells --- p.86 / Chapter III.2.2. --- Con A translocated the Protein Kinase C from Cytosol into Subnuclear Region --- p.86 / Chapter III.2.3. --- The Cell Death Induced by Con A Is Partially Inhibited by PKC Depletion But not by Staurosporine --- p.89 / Chapter Section 4 --- Discussions / Chapter I. --- PU5-1.8 cells as a Model for the Study of Cell Deathin Macrophages --- p.94 / Chapter II. --- Concanavalin A caused Cell Death in PU5-1.8 cells --- p.95 / Chapter III. --- Concanavalin A induced Programmed Cell Death in PU5-1.8 cells --- p.97 / Chapter IV. --- Increase in Intracellular Calcium was not Required in Con A-induced Cell Death --- p.100 / Chapter V. --- Activation of Protein Kinase C was Partially Required for Con A-induced Cell Death --- p.101 / Chapter VI. --- General Discussions --- p.102 / Chapter Section 5 --- Bibliography --- p.104 / Reference --- p.104

AIMS: The rapidly growing industrial and medical use of nanomaterials, especially zinc oxide and titanium dioxide, has led to growing concerns about their toxicity. Accordingly, the intrinsic genotoxic and cytotoxic potential of these nanoparticles have been evaluated. MATERIALS & METHODS: Using a HEp-2 cell line, cytotoxicity was tested along with mitochondrial activity and neutral red uptake assays. The genotoxic potential was determined using the Comet and the cytokinesis-blocked micronucleus assays. In addition, tyrosine phosphorylation events were investigated. RESULTS & CONCLUSION: We found concentration- and time-dependent cytotoxicity and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Our results suggest that both types of nanoparticles can be genotoxic over a range of concentrations without being cytotoxic.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.