Plasma and Nanomaterials: Fabrication and Biomedical Applications.

Application of plasma medicine has been actively explored during last several years. Treating every type of cancer remains a difficult task for medical personnel due to the wide variety of cancer cell selectivity. Research in advanced plasma physics has led to the development of different types of non-thermal plasma devices, such as plasma jets, and dielectric barrier discharges. Non-thermal plasma generates many charged particles and reactive species when brought into contact with biological samples. The main constituents include reactive nitrogen species, reactive oxygen species, and plasma ultra-violets. These species can be applied to synthesize biologically important nanomaterials or can be used with nanomaterials for various kinds of biomedical applications to improve human health. This review reports recent updates on plasma-based synthesis of biologically important nanomaterials and synergy of plasma with nanomaterials for various kind of biological applications.This work was supported by a grant from the National Research Foundation of Korea (NRF), which is funded by the Korean Government, Ministry of Science, ICT and Future Planning (MSIP) NRF-2016K1A4A3914113; NRF-2016R1C1B2010851 and also by the Ministry of trade; industry & energy grant No. 20131610101840. This work is also funded by Kwangwoon University in 2018-19

Identification of Genes Involved in Hematopoietic Stem Cell Differentiation and Leukemia Differentiation and Leukemia

Hematopoiesis is maintained by a proper balance between self renewal and multipotent differentiation of the hematopoietic stem cells (HSC). Acute myelogenous leukemia (AML) is characterized by the blockage in the differentiation of HSC, while self renewal and proliferation is preserved. It is important to understand the mechanisms involved in the inhibition of hematopoietic differentiation and maintenance of the HSC state in order to develop better therapies for AML. In these studies I have explored the role of Hsp90, omega-3 fatty acids and YB-1 in hematopoietic differentiation. EML, a hematopoietic precursor cell line, was used as a model for the hematopoietic system in these studies. My preliminary data showed the activation of Wnt signaling upon inhibition of Hsp90 in EML cells. This data suggested the involvement of Hsp90 in the regulation of Wnt signaling in EML cells. Moreover, my initial data with fatty acid studies indicated that omega-3 fatty acids could affect Wnt signaling in EML cells. Unfortunately, further progression of both these studies was marred by variability in my data. In my latest study, I have identified YB-1 as a marker involved in the maintenance of the hematopoietic stem cell state. YB-1 was found to be highly expressed in the EML cell line and in the mouse bone marrow-derived HSC and myeloid progenitor cells. In addition, YB-1 expression was downregulated during myeloid differentiation in retinoic acid (RA) and granulocyte macrophage colony stimulating factor (GM-CSF) treated EML cells, as well as in the granulocytes derived from mouse bone marrow. Further, abnormal YB-1 expression was observed in myeloid leukemic cell lines. Knockdown of YB-1 expression and arsenic trioxide treatment (As2O3) in erythroleukemic, K562 cell line resulted in apoptosis and inhibition of cell proliferation. Most importantly, these treatments led to the induction of megakaryocytic differentiation in these cells. Overall my data suggests that increased expression of YB-1 in the leukemic cells contributes to the leukemic cell properties by promoting cell proliferation, cell survival and blocking cell differentiation. Thus, YB-1 could be a potential target for therapy in myeloid leukemia

Transcriptional control of cellular plasticity in cancer cell senescence

Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern.Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype

Novel roles of KAT2A chromatin complexes ATAC and SAGA in normal and malignant haematopoiesis

Haematopoiesis is a dynamic process by which the full array of blood cell types originates from multipotent haematopoietic stem cells (HSCs). The blood system has long been an attractive model to explore how cell fate decisions are conducted at the molecular level, to generate the vast diversity of blood cell types. Blood cell fate choice is for the most part regulated by transcription factors and epigenetic modifiers. The enzymatic activities of epigenetic regulators are of particular importance in malignant haematopoiesis such as leukaemia, given their reversible nature and potential for therapeutic targeting. Epigenetic histone modifiers are often found within large multi-subunit complexes, whereby their catalytic activity is exercised. However, very little is known about how epigenetic enzymes take advantage of their participating chromatin complexes in order to accomplish their functions. In the blood system, no studies have formally examined how different epigenetic complexes with the same enzymatic component coordinate cell fate choices. Here, I focus on KAT2A, a lysine acetyltransferase responsible for Histone 3 Lysine 9 acetylation (H3K9ac), and a vulnerability in Acute Myeloid Leukaemia (AML), as well as its 2 containing complexes: Ada-Two-A-Containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). I combine cell functional and molecular assays of human cord blood (CB) haematopoietic cells and AML cells to dissect KAT2A roles depending on its participation in ATAC or SAGA complexes. Firstly, I demonstrate that KAT2A regulates CB erythroid progenitor specification and survival. I uncover unique contributions of the ATAC complex to erythroid lineage specification, whereas SAGA acts late in differentiation. This suggests that KAT2A plays stage-specific roles, which are unique to the complexes it integrates. While KAT2A regulates specification and survival of erythroid progenitors via ATAC through control of biosynthetic activity, it fine-tunes progression of erythroid differentiation through participation in SAGA. Secondly, I explored the roles of ATAC and SAGA in leukaemic haematopoiesis. I found that ATAC regulates proliferation through control of biosynthetic activity. Instead, SAGA participates in maintenance of the characteristic block in AML cell differentiation, compatible with a generic control of cell identity through preservation of cell-type specific transcriptional programmes. My data is consistent with a model in which the ATAC complex has pervasive roles in maintenance of self-renewing cells, while SAGA acts to stabilise cell-type specific programmes, preserving cell identity. Understanding ATAC and SAGA unique chromatin regulatory mechanisms has important implications for the tailored design of new drugs for clinical application.Rosetrees Trust PhD Studentship Academic Merit Scholarship - Santander Universities UK The Sir Richard Stapley Educational Trus

Engineered nanomaterials for biomedical applications

Engineered nanomaterials (ENM) have emerged as attractive and promising candidates for a wide range of advanced applications including in particular in medicine. However, the increased development of ENM raises the need to carefully assess their potential impact on human health and environment. For that, detailed evaluation of the intrinsic and biological identity of ENM is required for the safe design and use of these materials. To this effect, the present thesis focuses on the synthesis and biocompatibility assessment of two different classes of nanomaterials, dendrimers and superparamagnetic iron oxide nanoparticles (SPIONs), promising future nanomedicines for drug delivery and imaging agents in magnetic resonance imaging (MRI). Assessment was performed on primary human monocyte derived macrophages (HMDM), primary human bronchial epithelial cells (PBEC), and cell lines. Hereby an insight on the impact of these materials on the immune system and on their promising and potential use as nanomedicines has been obtained. Furthermore, we attempted to use systems biology approaches as a novel tool to identify possible hazard of ENM by using next generation sequencing RNA-Seq and computational tools. Finally, we assessed the bio-nano-interactions by evaluating the effect of the protein corona on the targeting capabilities of ENM and their behaviour. Importantly, the ENM were extensively characterized, using different techniques prior to the toxicity studies. In Paper I, we evaluated the biocompatibility of a library of polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) including dendrimers with two different surface functionalization, hydroxyl and carboxylic end groups, and two commercial polyamidoamine dendrimers (PAMAM) with amine and hydroxyl end groups. We found excellent biocompatibility for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose and time dependent cytotoxicity in the cell models tested. In paper II, using system biology approaches and bioinformatics tools, we were able to identify and validate the toxicity mechanism of PBEC exposed to PAMAMs dendrimers at low doses. Our studies showed that PAMAM-NH2, but not PAMAM-OH, caused down-regulation of cell cycle-related genes and cell cycle arrest in Sphase. Our findings provide evidence of the beneficial use of these new toxicology tools for the future risk assessment of nanomaterials. SPIONs have emerged as promising nanomaterials for biomedical applications, due to their excellent magnetic properties, chemical stability and biocompatibility. In paper III, ultrasmall superparamagnetic iron oxide nanoparticles (USIRONs) were prepared by a one-pot aqueous approach by using Fe(OH)3 as iron precursor, vitamin C as reducing agent, and dehydroascorbic acid (DHAA) as capping agent. We showed that USIRONs present high crystallinity, long-term colloidal stability, enhanced saturation magnetization, and exhibit excellent biocompatibility as demonstrated in the toxicity evaluation using primary HMDM. When nanoparticles are in contact with physiological fluids, adsorption of proteins on the surface of the nanomaterial will occur, resulting in the establishment of aprotein corona. Whether the protein corona will affect the targeting capabilities of the ENM was investigated. In paper IV, folic acid (FA)-conjugated iron oxide nanoparticles with poly(ethylene glycol) (PEG) or SiO2 surface coatings were synthetized. We evaluated their biocompatibility and specific targeting effects on HMDM and on ovarian cancer cells, that over express the folic acid receptor. Notably, we demonstrated the nanoparticles (NPs) were nontoxic to cells and that FA specific uptake was observed only for the FA iron oxide SiO2 coated NPs in the presence of serum proteins. Our studies contribute to the development of new nanomaterials and their applications, which may facilitate the clinical translation of the nanomedicines

Protein kinase CK2: analysis of its role in acute myeloid leukemia and conditional KO in mouse hematopoiesis

Hematopoiesis is a tightly controlled, hierarchically organized developmental process whereby hematopoietic stem cells (HSC) give rise to highly specialized, differentiated blood cells. HSC are characterized by the capacity of both self-renewing and differentiating in downstream committed hematopoietic progenitor cells. In vertebrates, hematopoiesis in embryonic and fetal life occurs in primitive hematopoietic organs whereas in the adult organism the definitive site of blood cell production is the bone marrow (BM). HSC are quiescent cells that reside most of the time in G0-G1 phases of the cell cycle, dividing during their lifespan as much as it is needed to maintain their own pool and give rise to committed precursors. Leukemia formation is characterized by the block of this process and accumulation of immature cells in the bone marrow and peripheral blood. In particular, acute myeloid leukaemia (AML) is characterized by the accumulation of large numbers of abnormal cells that fail to differentiate into functional mature blood cells. Leukemic blasts have a limited, exhaustible proliferative potential, suggesting that, in order to maintain leukemia growth, a small subpopulation of leukemic stem cells (LSCs) must display an inexhaustible proliferative capacity and self-renewal potential. No definitive proof of LSCs was available, however, until Dick and colleagues showed that the engraftment of NOD/SCID mice with primary AML samples could only be accomplished using cells that were phenotypically similar to normal hematopoietic stem cells (HSCs) by expressing CD34 and lacking markers of lineage commitment such as CD38 . HSCs and LSCs share common features: self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. The pathway regulating these functional properties can be categorized into self-renewal, developmental, and miscellaneous pathways, each of which is governed by a distinct set of critical genes that have emerged from molecular profiling and can be associated with stemness. In particular Wingless (Wnt)/β catenin and PI3K/AKT pathways are crucial for the control of both HSCs and LSCs biology, because they regulate proliferation, self renewal and differentiation of HSCs and they are involved in maintenance of LSCs. Phosphorylation is one of the molecular mechanism responsible for the signal transduction modulation of these two pathways and some molecules belonging to these pathways are phosphorylated by serin-threonin kinase CK2. Thus, this protein can have an important role in the biology of both HSCs and LSCs. CK2 is a pleiotropic and constitutive activated kinase, which has a tetrameric structure composed by two alpha catalytic subunits and two beta regulatory subunits, but these two components can work also separately. CK2 is essential for cell survival and proliferation, and is more and more evident its involvement in various aspects of tumor transformation. High levels of CK2 are found in different tumor type such as breast, lung, kidney cancer and also blood cancer. CK2 promotes the activity of proto-oncogenes (c-myc, C-Jun, A-Raf), drives cell cycle progression, stimulates beta-catenin activity, inhibits the onco-suppressors p53, PML and PTEN, and it can exert an anti-apoptotic effect through the inhibition of caspase activity. For this reasons, the principal aim of our research project is obtaining experimental evidence of a role of CK2 in normal hematopoiesis and in LSCs through the analysis of AML cell lines and samples collected from patients, and of a conditional knockout mouse model for CK2β in the hematopoietic system. As far as the role of CK2 in the hematopoiesis is concerned, our conditional KO mouse model for CK2β in the hematopoietic compartment, demonstrate that the regulatory subunit of CK2 has an important role in hematopoiesis. In particular, the KO of CK2β induces an impairment in hematopoiesis, especially in erythropoiesis. However, CK2β KO seems not to influence the HSCs pool but instead the hematopoietic progenitors. As far as the analysis of LSCs from AML samples, our analysis performed both on AML samples derive from patients and on Kasumi-1 cell line demonstrated that CK2 does not affect the Wnt/ β catenin and HH pathways, whereas it is able to modulate the PI3K/AKT pathway. Moreover, we have demonstrated that CK2 is important in LSCs survival as its inhibition increases the apoptosis and potentiates the effects of Daunorubicin, a drug currently used for AML treatment in clinic. Taken together, all our results indicate that CK2 possesses an important role both in hematopoiesis and in the biology of LSCs

How toxic are gold nanoparticles? The state-of-the-art.

With the growing interest in biotechnological applications of gold nanoparticles and their effects exerted on the body, the possible toxicity is becoming an increasingly important issue. Numerous investigations carried out, in the last few years, under different experimental conditions, following different protocols, have produced in part conflicting results which have leaded to different views about the effective gold nanoparticle safety in human applications. This work is intended to provide an overview on the most recent experimental results in order to summarize the current state-of-the-art. However, rather than to present a comprehensive review of the available literature in this field, that, among other things, is really huge, we have selected some representative examples of both in vivo and in vitro investigations, with the aim of offering a scenario from which clearly emerges the need of an urgent and impelling standardization of the experimental protocols. To date, despite the great potential, the safety of gold nanoparticles is highly controversial and important concerns have been raised with the need to be properly addressed. Factors such as shape, size, surface charge, surface coating and surface functionalization are expected to influence interactions with biological systems at different extents, with different outcomes, as far as gold nanoparticle potentiality in biomedical applications is concerned. Moreover, despite the continuous attempt to establish a correlation between structure and interactions with biological systems, we are still far from assessing the toxicological profile of gold nanoparticles in an unquestionable manner. This review is intended to provide a contribution in this direction, offering some suggestions in order to reach the systematization of data over the most relevant physico-chemical parameters, which govern and control toxicity, at different cellular and organismal levels

Modulatory effects of tryptanthrin on the murine myeloid leukemia cells.

Chan, Hoi Ling.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.Includes bibliographical references (leaves 206-220).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.iABBREVIATIONS --- p.iiABSTRACT --- p.viii撮要 --- p.xiiPUBLICATIONS --- p.xivTABLE OF CONTENTS --- p.xvChapter CHAPTER 1: --- GENERAL INTRODUCTIONChapter 1.1 --- Hematopoiesis & Leukemia --- p.1Chapter 1.1.1 --- An Overview on Hematopoiesis Development --- p.1Chapter 1.1.2 --- Leukemia --- p.6Chapter 1.1.2.1 --- General Symptoms of Leukemia --- p.7Chapter 1.1.2.2 --- Classification of Leukemia --- p.8Chapter 1.1.2.3 --- Conventional Treatment against Leukemia --- p.15Chapter 1.1.2.4 --- Novel Approaches --- p.19Chapter 1.2 --- The Chinese Medicinal Herb-Banlangen (板藍根） --- p.24Chapter 1.2.1 --- An Overview on Natural Indigo Compounds Derived from Banlangen --- p.24Chapter 1.2.2 --- Tryptanthrin --- p.29Chapter 1.2.2.1 --- Anti-bacterial Activity of Tryptanthrin --- p.29Chapter 1.2.2.2 --- Anti-tumor Activity of Tryptanthrin --- p.31Chapter 1.2.2.3 --- Anti-inflammatory Activity of Tryptanthrin --- p.33Chapter 1.2.2.4 --- Cutting Edges of Tryptanthrin as a Drug --- p.34Chapter 1.2.2.5 --- Metabolism of Tryptanthrin --- p.35Chapter 1.3 --- Aims and Scopes of This Investigation --- p.37Chapter CHAPTER 2: --- MATERIALS AND METHODSChapter 2.1 --- Materials --- p.39Chapter 2.1.1 --- Animals --- p.39Chapter 2.1.2 --- Cell Lines --- p.39Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.41Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.45Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.46Chapter 2.1.6 --- Reagents for DNA Extraction --- p.49Chapter 2.1.7 --- Reagents for Measuring Caspase Activity --- p.50Chapter 2.1.8 --- Reagents for Total RNA Isolation --- p.53Chapter 2.1.9 --- Reagents and Buffers for Reversed Transcription-PCR --- p.54Chapter 2.1.10 --- Reagents and Buffers for Real Time-PCR --- p.59Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.59Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.61Chapter 2.2 --- Methods --- p.70Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.70Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.70Chapter 2.2.3 --- Determination of Cell Viability --- p.71Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.72Chapter 2.2.5 --- Determination of Anti-leukemia Activity In Vivo --- p.73Chapter 2.2.6 --- Analysis of Cell Cycle Profile/DNA Content by Flow Cytometry --- p.74Chapter 2.2.7 --- Measurement of Apoptosis --- p.75Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.77Chapter 2.2.9 --- Measurement of Caspase Activity --- p.78Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species --- p.79Chapter 2.2.11 --- Gene Expression Study --- p.80Chapter 2.2.12 --- Protein Expression Study --- p.83Chapter 2.2.13 --- Measurement of Cell Differentiation --- p.87Chapter CHAPTER 3: --- STUDIES ON THE ANTI-PROLIFERATIVE EFFECT OF TRYPTANTHRIN AND INDIRUBIN-3'-OXIME ON MYELOID LEUKEMIA CELLSChapter 3.1 --- Introduction --- p.90Chapter 3.2 --- Results --- p.94Chapter 3.2.1 --- Effects of Indirubin-3'-oxime and Tryptanthrin on the Proliferation of Myeloid Leukemia Cell Lines of Human and Murine Origins In Vitro --- p.94Chapter 3.2.2 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.108Chapter 3.2.3 --- Cytotoxic Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.113Chapter 3.2.4 --- Cytotoxicity of Tryptanthrin on Non-Cancer Cell Line and Primary Myeloid Cells In Vitro --- p.115Chapter 3.2.5 --- Effects of Tryptanthrin on the Cell Cycle Profile of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.118Chapter 3.2.6 --- Effects of Tryptanthrin on the Expression of Cell Cycle Related Genes in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.123Chapter 3.2.7 --- Expression of CDK-inhibitors in Tryptanthrin-treated Murine Myeloid Leukemia WEHI-3B JCS Cells --- p.126Chapter 3.2.8 --- Effects of Tryptanthrin on the In Vivo Tumorigenicity of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.128Chapter 3.2.9 --- In Vivo Anti-tumor Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.130Chapter 3.3 --- Discussion --- p.132Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF TRYPTANTHRIN ON MURINE MYELOMONOCYTIC LEUKEMIA WEHI-3B JCS CELLSChapter 4.1 --- Introduction --- p.139Chapter 4.2 --- Results --- p.143Chapter 4.2.1 --- Induction of DNA Fragmentation by Tryptanthrin in the Murine Myelomonocytic Leukemia WEHI-3B Cells In Vitro --- p.143Chapter 4.2.2 --- Induction of Phosphatidylserine Externalization by Tryptanthrin in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.145Chapter 4.2.3 --- Change of Mitochondrial Membrane Potential of Tryptanthrin- treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.147Chapter 4.2.4 --- Induction of Caspase Activity in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.150Chapter 4.2.5 --- Induction of Reactive Oxygen Species in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.155Chapter 4.2.6 --- Expression of Bcl-2 Family Proteins in the Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.160Chapter 4.2.7 --- Effects of Tryptanthrin on the mRNA Expression of Bcl-2 Family Proteins in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.163Chapter 4.2.8 --- Expression of Fas and Fas Ligand Proteins in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.167Chapter 4.2.9 --- Expression of Pro-Apoptotic Protein in Tryptanthrin- treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.170Chapter 4.2 --- Discussion --- p.173Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ABILITY OF TRYPTANTHRIN ON MURINE MYELOMONOCYTIC LEUKEMIA WEHI-3B JCS CELLSChapter 5.1 --- Introduction --- p.184Chapter 5.2 --- Results --- p.186Chapter 5.2.1 --- Morphological Studies on Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.186Chapter 5.2.2 --- Effects of Tryptanthrin on the Cell Size and Granularity of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.189Chapter 5.2.3 --- Effects of Tryptanthrin on Induction of NBT-reducing Activity in the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.191Chapter 5.3 --- Discussion --- p.195Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.198REFERENCES --- p.20