by Poon Hong Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-161). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT (ENGLISH/CHINESE) --- p.ii / ABBREVIATIONS --- p.vi / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.viii / TABLE OF CONTENTS --- p.x / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.4 / Chapter I. --- Models for studying photoreceptor cell death --- p.4 / Chapter II. --- Photic retinopathy --- p.5 / Chapter II.1. --- Proposed Mechanisms of Photic Retinopathy --- p.5 / Chapter II.2. --- Factors Affecting Photic Retinopathy --- p.6 / Chapter II.3. --- Pathologic Processes of Photic Retinopathy --- p.7 / Chapter III. --- P53 --- p.9 / Chapter III.1. --- Historical Perspective --- p.9 / Chapter III.2. --- Structure of p53 --- p.10 / Chapter III.2.1. --- The p53 Gene & mRNA --- p.10 / Chapter III.2.2. --- The p53 Protein --- p.10 / Chapter III.3. --- Modifications of p53 Protein --- p.13 / Chapter III.4. --- Functions of p53 --- p.14 / Chapter III.4.1. --- p53 & Tumors --- p.14 / Chapter III.4.2. --- p53 & DNA Damage --- p.15 / Chapter III.4.3. --- p53 & Apoptosis --- p.16 / Chapter III.4.3.1. --- p53-dependent Apoptosis --- p.16 / Chapter III.4.3.2. --- p53-independent Apoptosis --- p.20 / Chapter IV. --- clusterin --- p.22 / Chapter IV. 1. --- Historical Perspective --- p.22 / Chapter IV.2. --- Structure of Clusterin --- p.22 / Chapter IV.2.1. --- Clusterin Gene & mRNA --- p.22 / Chapter IV.2.2. --- Clusterin Protein --- p.22 / Chapter IV.3. --- Functions of Clusterin --- p.22 / Chapter IV.3.1. --- Clusterin & Apoptosis --- p.23 / OBJECTIVES --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter V. --- Sample collections --- p.26 / Chapter V.l. --- Induction of Photic Injury in Rat --- p.26 / Chapter V.2. --- Tissue Processing --- p.26 / Chapter V.2.1. --- Paraffin-embedded Tissues --- p.26 / Chapter V.2.2. --- Epoxy-embedded Tissues --- p.27 / Chapter V.3. --- Cell Culture --- p.28 / Chapter VI. --- Light-microscopic study --- p.29 / Chapter VI. 1. --- Histopathology --- p.29 / Chapter VI.1.1. --- Toluidine Blue --- p.29 / Chapter VI. 1.2. --- Morphometry of the ONL Thickness --- p.29 / Chapter VI.2. --- In situ TUNEL --- p.29 / Chapter VI.2.1. --- Morphometry of TUNEL --- p.29 / Chapter VI.3. --- Immunohistochemistry --- p.30 / Chapter VI.3.1. --- Single-labeling --- p.30 / Chapter VI.3.1.1. --- Immunolabeling of p53 Protein --- p.31 / Chapter VI.3.1.2. --- Immunolabeling of p21 Protein --- p.31 / Chapter VI.3.1.3. --- Immunolabeling of bax Protein --- p.31 / Chapter VI.3.1.4. --- Immunolabeling of c-fos Protein --- p.32 / Chapter VI.3.1.5 --- Immunolabeling of Clusterin Protein --- p.32 / Chapter VI.3.2. --- Double-labeling --- p.32 / Chapter VI.3.2.1. --- TUNEL & Fluorescent-labeling of p53 --- p.32 / Chapter VI.3.2.2. --- TUNEL & Fluorescent-labeling of p21 --- p.33 / Chapter VI.3.3. --- Grading of Immunoreactivities --- p.33 / Chapter VI.3.4. --- Morphometry of c-fos Immuno-positive Nuclei --- p.33 / Chapter VI.4. --- In situ RT-PCR --- p.34 / Chapter VI.4.1. --- Isolation of Retinal DNA --- p.34 / Chapter VI.4.2. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter VI.4.3. --- In situ Reverse Transcriptase 226}0ؤ PCR (RT-PCR) --- p.35 / RESULTS --- p.37 / Chapter VII. --- LIGHT-MICROSCOPY --- p.37 / Chapter VII.1. --- Histopathology & Morphometry --- p.37 / Chapter VII. 1.1. --- Histopathology --- p.37 / Chapter VII.1.2. --- Morphometry of the ONL Thickness --- p.49 / Chapter VII.2. --- DNA Nicks Detection --- p.42 / Chapter VII.2.1. --- In situ TUNEL --- p.42 / Chapter VII.2.2. --- Morphometry of TUNEL --- p.42 / Chapter VII.3. --- p53 --- p.51 / Chapter VII.3.1. --- Immunohistochemistry of p53 --- p.51 / Chapter VII.3.2. --- Grading of p53 Immunoreactivities --- p.51 / Chapter VII.3.3. --- p53 in situ RT-PCR --- p.51 / Chapter VII.3.4. --- Double-labeling ofp53 with in situ TUNEL --- p.59 / Chapter VII.4. --- p21 --- p.74 / Chapter VII.4.1. --- Immunohistochemistry of p21 --- p.74 / Chapter VII.4.2. --- Grading of p21 Immunoreactivities --- p.74 / Chapter VII.4.3. --- Double-labeling of p21 with in situ TUNEL --- p.74 / Chapter VII.5. --- bax --- p.84 / Chapter VII.5.1. --- Immunohistochemistry of bax --- p.84 / Chapter VII.5.2. --- Grading of bax Immunoreactivities --- p.84 / Chapter VII.5.3. --- bax in situ RT-PCR --- p.84 / Chapter VII.6. --- c-fos --- p.96 / Chapter VII.6.1. --- Immunohistochemistry of c-fos --- p.96 / Chapter VII.6.2. --- Morphometry of c-fos immuno-positive nuclei & TUNEL --- p.96 / Chapter VII.7. --- Clusterin / Chapter VII.7.1. --- Immunohistochemistry of Clusterin --- p.108 / Chapter VII.7.2. --- Grading of Clusterin Immunoreactivities --- p.108 / discussion --- p.116 / Chapter VIII. 1. --- Summary --- p.116 / Chapter VIII.1.1. --- Histopathology & Morphometry --- p.118 / Chapter VIII.1.2. --- In situ TUNEL --- p.119 / Chapter VIII.1.3. --- p53 & c-fos --- p.119 / Chapter VIII.1.4. --- p27 --- p.124 / Chapter VIII.1.5. --- bax --- p.124 / Chapter VIII. 1.6. --- Clusterin --- p.124 / Chapter VIII.2. --- Hypothesis --- p.125 / Chapter VIII.3. --- Conclusion --- p.127 / bibliography --- p.129

Programmed cell death (PCD) is an essential biological process in animal development and tissue homeostasis that is necessary to ensure the physiological well-being of the organism. During PCD, phagocytes facilitate the selective removal of excess, damaged, and potentially deleterious cells, in a multi-step engulfment process. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signal transduction pathways that act redundantly to regulate engulfment: the CED-1/-6/-7 and CED-2/-5/-12 pathways. Of these cell death (CED) proteins, the ABC transporter CED-7 is the only protein reported to be required in both the engulfing cell and the dying cell. However, its function in the cell death process remains the most enigmatic and the ced-7 ortholog previously has not been identified in Drosophila. Homology searches revealed a family of putative ced-7 orthologs that encode transporters of the ABCA family in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1 in PCD, we analyzed their engulfment function in oogenesis, during which 15 germ cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. It has been shown that genetically knocking down individual engulfment genes results in inefficient clearance of the germ cells, which then persist in late-stage egg chambers. Only two of the putative ced-7/ABCA1 genes are expressed significantly in the ovary, CG31731 and CG1718, and we have characterized these genes using transposon insertions, deficiencies, and RNAi knockdowns. Our genetic analysis reveals that CG31731 is necessary for germ cell clearance in the Drosophila ovary. Immunostaining shows that genetically knocking down CG31731 results in uncleared germ cells which persist in late-stage egg chambers. Altogether, our findings suggest that CED-7/ABCA1/CG31731 play evolutionarily conserved roles during engulfment.

The neonatal subventricular zone (SVZ) is a site of continued postnatal neurogenesis, and is the source of cortical glial cells. Apoptosis is an endogenous process of cell destruction, and is a key event in the proper development of the SVZ. Despite its importance, there is still a lack of knowledge regarding the temporal and spatial occurrence of neonatal SVZ apoptosis, cell types affected and the underlying intrinsic and extrinsic mechanisms that guide the process. This thesis addresses these issues, and in addition, finds a nontraditional mode of neurotrophic action for cell survival in the neonatal SVZ. We assessed SVZ apoptosis by subregion, employing the cell death markers, pH2ax and cleaved caspase 3. The medial SVZ contained the highest density of dying cells at p0, while at p7 there was no significant difference in the apoptotic cell density distribution in the SVZ subregions. Combining cell type specific markers with the death markers used, revealed immature postmitotic neurons were the primary cell type cleared in the p0 medial SVZ. The majority of dying cells in the p7 dorsolateral SVZ (SVZdl) were unable to be identified. Using stereotactic injection of a GFP expressing lentivirus, we determined the p0 medial SVZ cell population to be migratory cells bound for the olfactory bulb. An investigation into the intrinsic and extrinsic mechanisms mediating cell death in the neonatal SVZ, showed BH3-only protein Bim expression in the p0 and p7 SVZ, as well as significantly decreased p0 medial SVZ apoptosis in Bim knockout mice. Bim knockout mice did not show a significant change in apoptosis in the p7 SVZdl. TrkB knockout mice have shown a survival role for the receptor in the lateral ganglionic eminence of the neonatal SVZ. To test this in the p0 medial SVZ using a more specific method, a TrkB blocking antibody was injected into the p0 medial SVZ. This resulted in a significantly higher number of apoptotic cells in the p1 medial SVZ versus controls. These studies demonstrate the dynamic nature of the SVZ with its changing density and identity of apoptotic cells within the subregions. It has also shown the influence of Bim and TrkB signaling in neonatal SVZ apoptosis and survival. Finally, it has identified a premigratory cell population in the p0 medial SVZ, whose survival is mediated by neurotrophin signaling at their site of origin.

Cells die not merely as a consequence of catastrophic failure of homeostasis but when programmed cell death is activated. The existence of non-apoptotic modes of regulated cell death is increasingly appreciated. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. In this thesis, we developed a systematic framework to discover and characterize lethal compounds that induce distinct cell death phenotypes. In the first part, we investigated the landscape of pharmacologically-accessible non-apoptotic cell death mechanisms. This effort resulted in the discovery of a novel ferroptosis inducer, FIN56. The rest of my work focused on characterizing the mechanism of action of FIN56. Technologies used here should be generally applicable for the systematic study of various cell death mechanisms. First, to globally survey pharmacologically-accessible cell death mechanisms, we used 3,169 uncharacterized lethal compounds as cell death probes. We found that 451 compounds (14%) were lethal without activating caspase activity. 56 most potent and structurally diverse compounds were more closely studied using the 'modulatory profiling' approach, which involves examining changes in potency of lethal compounds by co-treatment with chemical modulators. We discovered that caspase-independent lethals induced three types of regulated non-apoptotic cell death: metal ion-dependent cell death, necrostatin-1-dependent cell death, and ferroptosis, a regulated form of iron-dependent oxidative cell death. With further structural optimization, we discovered a specific ferroptosis inducer, FIN56. Ferroptosis is induced when the lipid repair enzyme glutathione peroxide 4 (GPX4) is inhibited or inactivated by depletion of glutathione. We found that, in contrast, FIN56 induced ferroptosis through decreasing the abundance of GPX4. Second, we developed a technology that identifies proteins responsible for cell death mechanisms of interest utilizing chemical library screening. The technology consists of three steps: (i) binding targets of each molecule in the chemical library were predicted using Similarity Ensemble Approach, a chemoinformatic ligand-based target prediction algorithm; (ii) the chemical library was screened for enhancers/suppressors of the cell death; (iii) incorporating the screening data into the prediction to make the prediction more reliable. This approach, termed `Target Enrichment Analysis', resulted in the discovery of two features of FIN56-induced ferroptosis: calcium ion influx and activation of lipoxygenases, enzymes that peroxidize fatty acids. Inhibiting either of them suppressed FIN56-induced ferroptosis. Third, we tried to capture metabolic changes induced upon FIN56 treatment that were relevant to the mechanism of action of FIN56 and identify protein targets of FIN56 using chemoproteomics. Metabolomic profiling experiments discovered that non-steroidogenic intermediates in the mevalonate pathway regulated cellular sensitivity to FIN56-induced ferroptosis. Although none of the proteins identified through target identification effort has yet been fully confirmed as responsible for induction of ferroptosis triggered by FIN56, we found through the analysis that inhibition of squalene synthase, an enzyme in the mevalonate pathway, suppressed FIN56-induced ferroptosis consistently. Finally, to define biomarkers that predict sensitivity to ferroptosis inducers including FIN56, we investigated the molecular determinants of sensitivity in the NCI60 panel and identified nicotinamide adenine dinucleotide phosphate (NADPH) levels as a global predictor of sensitivity to ferroptosis. These studies demonstrate that sensitivity to ferroptosis is regulated by metabolic pathways, suggesting that it may be a relevant form of cell death in cases of dysregulated metabolism. This systematic approach using a combination of modulatory profiling and cell line selectivity analysis is an effective means to explore, discover and characterize cellular phenotypes induced by unknown small molecules.

Every year, in the United States alone, there are 1.7 million incidences of traumatic brain injury (TBI). Unfortunately, despite the tremendous societal and economic cost and decades of research, current pharmacological treatments for TBI are lacking. The specific aims of this thesis are: (1) to determine the efficacy of 17β-estradiol (E2) monotherapy treatment post-TBI, (2) determine if a combination treatment of E2 and memantine provides statistically significant benefits over monotherapy treatments post-TBI, and (3) to investigate the utility of an in vitro model to recapitulate the pathobiology of an in vivo model of TBI and to assess its potential to discover novel and clinically relevant therapeutic targets for future studies. The neuroprotective properties of E2 have been investigated for several decades in several different models including excitotoxicity, ischemia, and TBI. Organotypic hippocampal slice cultures (OHSCs) were mechanically injured at specified strain and strain rates which are relevant to TBI, and the efficacy of E2 post-TBI was investigated. Physiological concentrations of E2 were more effective at preventing cell death than supraphysiological concentrations. Further, GPR30, a novel G protein-coupled receptor, was not activated at physiological concentrations. These results suggest that the classical estrogen receptors (ERs) were primarily responsible for E2-mediated neuroprotection following TBI, and that GPR30 is neither necessary nor sufficient. While monotherapy treatments have shown preclinical success post-TBI, none have been successful in clinical trials. Combination therapies are a promising area of research that focuses on synergistic effects between compounds for significant increases in neuroprotection, potentially resulting in a clinically relevant treatment. A combination treatment of E2 and memantine was statistically more neuroprotective than either monotherapy post-TBI. Using micro-electrode arrays (MEAs), we recorded and quantified increased evoked responses in OHSCs after physiological concentrations of E2 and showed that memantine significantly reduces these effects. Our results suggest a potential combination treatment for TBI and a possible mechanism for its synergistic effects. TBI is a complex injury which initiates a multitude of secondary injuries causing delayed cell death for days or beyond. The utility of in vitro models depends on their ability to recapitulate the in vivo injury cascade after TBI. We used a genome wide approach to study changes in gene expression after injury in both an in vitro model and an in vivo model of TBI to compare the post-TBI pathobiology. There was a strong correlation in gene expression changes between the two models providing confidence that the in vitro model represented the in vivo injury cascade. From these data, we searched for genes with significant changes in expression over time and identified Sorla. Sorla directs amyloid precursor protein (APP) to the recycling pathway by direct binding and away from amyloid beta (Aβ) producing enzymes. Mutations of Sorla have been linked to Alzheimer's disease (AD). We confirmed the down regulation of SORLA expression in OHSCs by immunohistochemistry (IHC) and western blotting. Together, these data suggests that the in vitro model of TBI that was tested strongly recapitulates the in vivo TBI pathobiology and is well-suited for future mechanistic or therapeutic studies. The data also suggest a novel target, Sorla, which may play a role in AD caused by TBI. In conclusion, we discovered a potentially clinically relevant combination treatment of E2 and memantine for post-TBI therapy. We also confirmed that our in vitro model of TBI is well representative of in vivo models, and that relevant, novel targets for future TBI studies can be elucidated with this model. A potential link between AD and TBI was suggested and warrants future study. Together, these studies address the growing public health concern of TBI.

Cells die when they are starved of thymidine, one of the four DNA nucleotides. Since the discovery of this killing phenomenon, termed thymineless death (TLD), researchers have been trying to understand why. The goal of the work presented here is to use systems level approaches to shed light on this process. Because DNA synthesis is the only cellular process that requires thymidine, it is logical that the focus has been mainly on DNA stability and damage. My work expands the focus to new frontiers: acetate metabolism, the cytoplasm and the inner membrane. I generated thymidine auxotrophs in two genetic backgrounds by inactivating the thymidylate synthase enzyme, thyA. These mutants need supplementation with exogenous thymidine in order to survive. I used these strains in three experimental approaches to explore the mechanisms of TLD. Fitness profiling of a transposon insertion library in a thyA- strain, long-term laboratory evolution during thymidine-limitation, and RNA sequencing of TLD-sensitive and TLD-resistant strains identified genes in previously known processes as well as genes in novel processes. These approaches allowed me to gather rich data sets that identified many contributing genes. 52 genes showed consistent effects across approaches. My work confirms that ROS is a key contributor to killing during thymidine starvation and reveals that putrescine biosynthesis enzymes, an acetate overflow kinase, and the proton-transporting ATP synthase are novel players in TLD. I suggest that these three novel players contribute through their shared role in modulating cytoplasmic pH and propose a model in which DNA damage, ROS accumulation, and cytoplasmic acidification converge on the killing process during thymidine starvation. My findings expand the sites of critical action during TLD from the DNA to the cell’s inner and outer membranes and the cytoplasm. Theories on active vs. passive and specific vs. general bacterial death pathways will be discussed at the end.

The ability of organisms to sense and respond to challenges to their genome integrity is key to survival. In particular, the ability to detect and respond to double-stranded DNA breaks (DSBs) is of fundamental importance as not only are DSBs potentially lethal as they can trigger apoptosis, but there is also the potential for the loss of genetic information. The response to DSBs is well conserved across Eukaryotes and comprises two stages: detection of the break and subsequent remedial action. The remedial action involves cell cycle arrest, DNA repair, and, if repair cannot be effected, possible apoptosis. Whilst many of the key components, especially in the initial detection of the break, are conserved there are also differences between plants and animals in some of the main components and their roles. In this thesis I have proposed an overall framework for the cellular response to DSBs in plants and have proposed two candidate genes, TCP20 and SOG1, as novel plant specific activators in this response. Their suitability has been addressed by considering their activation and their downstream targets. I have shown that TCP20 is necessary for growth arrest observed in shoot apical meristems after exposure to genotoxic stress. I have also shown that activation of one of the key targets of TCP20, CYCB1;1 requires TCP20 and that a key TCP20 binding motif in the promoter of CYCB1;1 is necessary for the up-regulation of CYCB1;1 in response to genotoxic stress. This motif is over-represented in the promoters of many of the genes involved in DNA damage repair, suggesting that TCP20 plays a role in the co-ordination of the cellular response to DSBs.

by Lau Kwok Wing, Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 166-188). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 : --- GENERAL INTRODUCTION --- p.1 / Chapter (1.1) --- Hematopoiesis : An Overview --- p.2 / Chapter (1.1.1) --- Development of Blood Cells and Sites of Hematopoiesis --- p.2 / Chapter (1.1.2) --- Hematopoietic Cytokine Network --- p.4 / Chapter (1.1.3) --- Molecular Control of Hematopoietic Cell Development --- p.5 / Chapter (1.2) --- Leukemia : An Overview --- p.8 / Chapter (1.2.1) --- Leukemia : Abnormalities in Blood Cell Formation --- p.8 / Chapter (1.2.2) --- Pathophysiology and Etiology of Leukemia --- p.10 / Chapter (1.2.3) --- New Avenues for Therapy : Induction of Differentiation and Apoptosis --- p.12 / Chapter (1.3) --- Induction of Differentiation in Myeloid Leukemia Cells --- p.14 / Chapter (1.3.1) --- Inducers of Leukemic Cell Differentiation --- p.14 / Chapter (1.3.2) --- Cytokines as Inducers of Myeloid Leukemic Cell Differentiation --- p.17 / Chapter (1.3.3) --- Phenotypic Changes and Functional Characterizations --- p.20 / Chapter (1.3.4) --- Modulation of Gene Expression in Myeloid Leukemic Cell Differentiation --- p.22 / Chapter (1.4) --- Apoptosis and Leukemic Cell Death --- p.23 / Chapter (1.4.1) --- Apoptosis : An Overview --- p.23 / Chapter (1.4.2) --- Cytokines and Apoptosis in Myeloid Leukemia --- p.26 / Chapter (1.5) --- Objectives and Research Strategy --- p.27 / Chapter (1.5.1) --- The Murine Myeloid Leukemia Cell Line (Ml) as an Experimental Cell Model for Acute Myeloid Leukemia --- p.27 / Chapter (1.5.2) --- Leukemia Inhibitory Factor (LIF) as a Differentiation Inducer --- p.28 / Chapter (1.5.3) --- Aims and Scopes of This Investigation --- p.31 / Chapter CHAPTER 2 ： --- MATERIALS AND METHODS --- p.33 / Chapter (2.1) --- Materials --- p.34 / Chapter (2.1.1) --- Mice --- p.34 / Chapter (2.1.2) --- Cell Lines --- p.34 / Chapter (2.1.3) --- Recombinant Cytokines --- p.34 / Chapter (2.1.4) --- Monoclonal Antibodies --- p.36 / Chapter (2.1.5) --- Oligonucleotide Primers and Internal Probes --- p.37 / Chapter (2.1.6) --- "Buffers, Culture Medium and Other Reagents" --- p.39 / Chapter (2.1.7) --- Reagents and Solutions for Gene Expression Study --- p.41 / Chapter (2.2) --- Methods --- p.46 / Chapter (2.2.1) --- Culture of Myeloid Leukemia Cell Lines --- p.46 / Chapter (2.2.2) --- Induction of Leukemic Cell Differentiation --- p.46 / Chapter (2.2.3) --- Determination of Cell Growth and Proliferation --- p.46 / Chapter (2.2.4) --- Cell Morphological Study --- p.47 / Chapter (2.2.5) --- Assessment of Differentiation-Associated Characteristics --- p.48 / Chapter (2.2.5.1) --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter (2.2.5.2) --- Phagocytosis Assay --- p.48 / Chapter (2.2.5.3) --- Assay of Plastic Adherence --- p.49 / Chapter (2.2.6) --- Flow Cytometric Analysis --- p.49 / Chapter (2.2.6.1) --- Surface Antigen Immunophenotyping --- p.49 / Chapter (2.2.6.2) --- Assay of Endocytic Activity --- p.50 / Chapter (2.2.6.3) --- Assay of Non-specific Esterase Activity --- p.50 / Chapter (2.2.6.4) --- Cell Cycle / DNA Content Evaluation --- p.51 / Chapter (2.2.7) --- Gene Expression Analysis --- p.52 / Chapter (2.2.7.1) --- Preparation of Cell Lysate --- p.52 / Chapter (2.2.7.2) --- RNA Isolation --- p.52 / Chapter (2.2.7.3) --- Reverse Transcription --- p.53 / Chapter (2.2.7.4) --- Polymerase Chain Reaction (PGR) --- p.54 / Chapter (2.2.7.5) --- Agarose Gel Electrophoresis --- p.55 / Chapter (2.2.7.6) --- 3' End Labelling of Oligonucleotide Probes --- p.56 / Chapter (2.2.7.7) --- Dot Blot Hybridization --- p.56 / Chapter (2.2.7.8) --- Digoxigenin (DIG) Chemiluminescent Detection --- p.57 / Chapter (2.2.8) --- DNA Fragmentation Analysis --- p.58 / Chapter (2.2.9) --- Statistical Analysis --- p.59 / Chapter CHAPTER 3 ： --- "EFFECTS OF LEUKEMIA INHIBITORY FACTOR ON THE PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS OF MURINE MYELOID LEUKEMIA Ml CELLS" --- p.60 / Chapter (3.1) --- Introduction --- p.61 / Chapter (3.2) --- Results --- p.63 / Chapter (3.2.1) --- Induction of Growth Arrest in rmLIF-Treated Ml Cells --- p.63 / Chapter (3.2.2) --- Induction of Monocytic Differentiation of Ml cells by rmLIF --- p.66 / Chapter (3.2.2.1) --- Morphological Changes --- p.66 / Chapter (3.2.2.2) --- Induction of Plastic Adherence --- p.70 / Chapter (3.2.2.3) --- Surface Antigen Immunophenotyping --- p.70 / Chapter (3.2.2.4) --- NBT-Reducing Activity of rmLIF-Treated Ml Cells --- p.76 / Chapter (3.2.2.5) --- Non-specific Esterase Activity of rmLIF-Treated Ml Cells --- p.77 / Chapter (3.2.2.6) --- Endocytic Activity of rmLIF-Treated Ml Cells --- p.78 / Chapter (3.2.2.7) --- Phagocytic Activity of rmLIF-Treated Ml Cells --- p.79 / Chapter (3.2.3) --- Induction of Differentiation-Associated DNA Fragmentation --- p.80 / Chapter (3.2.4) --- Production of Differentiation-Inducing Factors --- p.84 / Chapter (3.3) --- Discussion --- p.88 / Chapter CHAPTER 4 : --- CYTOKINE INTERACTIONS IN REGULATING THE PROLIFERATION AND DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.94 / Chapter (4.1) --- Introduction --- p.95 / Chapter (4.2) --- Results --- p.97 / Chapter (4.2.1) --- Synergistic Effect of LIF and IL-6 on the Proliferation and Differentiation of Ml Cells --- p.97 / Chapter (4.2.2) --- Regulation of Proliferation and Differentiation of Ml Cells by LIF and OSM --- p.101 / Chapter (4.2.3) --- Effects of LIF and TNF-α on the Proliferation and Differentiation of Ml Cells --- p.104 / Chapter (4.2.4) --- Synergistic Effect of LIF and IL-1 on the Proliferation and Differentiation of Ml Cells --- p.107 / Chapter (4.3) --- Discussion --- p.115 / Chapter CHAPTER 5 ： --- MODULATION OF CYTOKINE AND CYTOKINE RECEPTOR GENE EXPRESSION IN LIF- INDUCED DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.120 / Chapter (5.1) --- Introduction --- p.121 / Chapter (5.2) --- Results --- p.123 / Chapter (5.3) --- Discussion --- p.152 / Chapter CHAPTER 6 ： --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.158 / REFERENCES --- p.166

No description available.

Both small molecules and antibodies are powerful tools for research in biological mechanisms and therapeutics. The discovery of such molecules involves two opposite starting points: one being specific targets and the other being phenotypic screens. The first part of this thesis focuses on drug development starting with a specific target. The second part of this thesis focuses on identification of ferroptosis biomarkers by phenotypic screen. The specific target highlighted in the first part of this thesis is KRAS (Kirsten rat sarcoma viral oncogene homolog), the most commonly mutated
 oncogene in human pancreatic cancers, colorectal cancers, and lung cancers. The high prevalence of KRAS
mutations and its prominent role in many cancers make it a
potentially attractive drug target; however, it has been difficult
 to design small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on
K-RasG12D, which we have termed the P110 site (due to its adjacency to proline 110), using computational analyses of the protein structure. We then confirmed that one compound, named K-Ras Allosteric Ligand KAL-21404358, might bind to the P110 site of K-RasG12D using a combination of computational
 and biochemical approaches. The phenotypic screen used in the second part of this thesis focus on the process of ferroptosis, a form of regulated cell death process driven by the iron-dependent accumulation of polyunsaturated-fatty-acid-containing phospholipids (PUFA-PLs). Currently, there is no way to selectively stain ferroptotic cells in tissue sections to characterize relevant models and diseases. To circumvent this problem, we immunized mice with membranes from diffuse large B Cell lymphoma (DLBCL) cells treated with piperazine erastin (PE), and screened the generated monoclonal antibodies. The results suggested that for the first time we could detect cells undergoing ferroptosis in human tissue sections. In summary, these two projects illustrate how molecular screening and design starting from either a specific target or a phenotype screen aid in drug and biomarker development.