As embryos proceed through development, they must undergo a series of cell fate decisions. At each division, potency is progressively restricted until a terminally differentiated, postmitotic cell is produced. An important part of that cell type determination is repression of alternative fate possibilities. In this thesis, I have explored the mechanisms by which a single transcription factor activates certain cell fates while inhibiting others, using the Caenorhabditis elegans ALM and BDU neurons as a model. ALM neuron identity is regulated by two interacting transcription factors: the POU homeobox gene unc-86 and the LIM homeobox gene mec-3. I investigated fate determination in BDU neurons, the sister cells of ALM. I found that BDU identity is broadly defined by a combination of unc-86 and the Zn finger transcription factor pag-3, while the neuropeptidergic subroutine of BDU is determined by the LIM homeobox gene ceh-14. In addition, I found that reciprocal homeotic transformations occur between ALM and BDU neurons upon loss of either mec-3 or pag-3. In mec-3 mutants, ALM neurons acquire the gene expression profile and morphological characteristics of BDU cells, while in pag-3 mutants, BDU neurons express genes normally found in ALM and change some aspects of their morphology to resemble ALM. While these fate switches appear to be a simple case of cross-repression, the mechanism is in fact more complicated, as pag-3 is expressed not just in BDU but also in ALM. In this thesis, I present evidence that MEC-3 inhibits execution of BDU identity in ALM by physically binding to UNC-86 and sequestering it away from the promoters of BDU genes. This work expands upon the literature examining simultaneous activation of one identity program and repression of alternate programs by introducing a novel mechanism by which a transcription factor competes to direct specific cell fates.

Dendritic cells (DCs) comprise a distinct lineage of potent antigen-presenting mononuclear phagocytes that serve as both mediators of innate immune responses and key facilitators of the adaptive immune response. DCs play both immunogenic and tolerogenic roles through their dual ability to elicit pathogen-specific T cell immunity as well as induce regulatory T cell (Treg) responses to promote tolerance in the steady state. The aim of the work presented here is to examine the normal regulatory mechanisms of DC development and function, starting with the dissection of mechanisms behind an aberrantly activated developmental pathway, followed by the exploration of new mechanisms governed by two candidate transcription factors. The first chapter of the thesis focuses on the growth factor receptor Flt3, an essential regulator of normal DC development in both mice and humans, and concurrently one of the most commonly mutated proteins found in acute myeloid leukemia (AML). We investigated the effect of its most common activating mutation in AML, the Flt3 internal tandem duplication (Flt3-ITD), and found that this mutation caused a significant cell-intrinsic expansion of all DC populations. This effect was associated with an expansion of Tregs and the ability to dampen self-reactivity, with an inability to control autoimmunity in the absence of Tregs. Thus, we describe a potential mechanism by which leukemia can modulate T cell responses and support Treg expansion indirectly through DCs, which may compromise immunosurveillance and promote leukemogenesis. The subsequent chapters explore the basic molecular mechanisms of DC development by using Flt3 expression as a guide to uncover new candidates involved in the DC transcriptional program. We show that Myc family transcription factor, Mycl1, is largely dispensable for DC development and function, contrary to recent published findings that propose a role in proliferation and T cell priming. On the other hand, we find that conditional deletion of our second candidate gene, an Ets family transcription factor, has diverse effects on DC development, monocyte homeostasis, and cytokine production. Overall, our studies highlight an unexpected molecular link between DC development and leukemogenesis, and elucidate novel mechanisms controlling DC differentiation and function.

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células β pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células β são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células β , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell

Doenças envolvendo órgãos endodermicamente derivados afetam milhares de pessoas no mundo. O sucesso da terapia celular para o tratamento de doenças de órgãos oriundos da mesoderme e ectoderme gera ótimas perspectivas para o uso do mesmo em tratamento de doenças de órgãos de origem endodérmica (tireóide, pulmões, fígado, vesícula biliar e pâncreas). Particularmente, no fígado, ainda que sejam atribuídas propriedades regenerativas em muitas lesões o mecanismo de reparação é insuficiente, tornando o transplante hepático à única opção definitiva. Dentre as células-tronco embrionárias, as células de linhagens hepáticas fetais podem estabelecer-se como fonte importante para a terapia celular em indivíduos com doenças hepáticas, pois possuem um alto índice de diferenciação de hepatócitos e células do ducto biliar in vitro. Evidências apontam a progranulina como um fator de crescimento de grande habilidade para a indução de proliferação celular, uma vez que está envolvida no desenvolvimento embrionário e neonatal. Sendo assim, neste trabalho foram utilizados embriões de ratos Fischer 344 com idades gestacionais 12,5; 13,5; 14,5; 15,5; 16;5 para caracterizar o papel do novo fator de crescimento progranulina na hepatogênese. Foram realizadas análises histológicas, de microscopia eletrônica de transmissão e imuno-histoquímicas nos embriões. Houve expressão de progranulina e Oct-4 (marcador de célula tronco indiferenciada) principalmente nas idades gestacionais de 13,5 a 16,6 dias e 12,5 a 16,5 dias para PCNA (marcador de proliferação celular). Dessa forma acredita-se que, a progranulina atua nos processos de proliferação celular e diferenciação das células-tronco do broto hepático, podendo ser usada como um fator de diferenciação em culturas in vitro visando à diferenciação de células-tronco do broto hepático em hepatócitos funcionais para a terapia celular. / Diseases involving endodermal-derivated organs affect thousands of people all over the world. The cell therapy success for treating diseases of organs originated from mesoderm and ectoderm generates excellent perspectives for its use in treating diseases of organs of endodermal origin (thyroid, lungs, liver, gallbladder and pancreas). Particularly in the liver, although regenerative proprieties are attributed in many lesions, the repairing mechanism is insufficient, making the hepatic transplant the only definitive option. Among the embryo stem cells, the fetal hepatic lineage cells may establish themselves as important sources for cell therapy in individuals with hepatic diseases, since they have high ability in hepatocytes and billiar duct cells differentiation in vitro. However, for the use of hepatic lineage embryo stem cells as a bi-potential source of differentiation, it is necessary the establishment of efficient proliferation methods in this type of cells. Evidences point to progranuline as a growth factor with high capability for the induction of cell proliferation, since it is involved in the embryo and neo-natal development. Thus, this study used embryos of Fischer 344 rats with gestational ages 12.5, 13.5, 14.5, 15.5 and 16.5 to characterize the role of the new growth factor progranuline in hepatogenesis. We conducted histological, transmission electron microscopy and immunohistochemical in embryos. There was expression of progranuline and Oct-4 (undifferentiated stem cell marker), especially at gestational ages 13.5 to 16.5 days and 12.5 to 16.5 days for PCNA (proliferation marker). So it is believed that progranuline acts on cell proliferation and differentiation of stem cells from the liver bud, which can be used as a differentiating factor in order to cultures in vitro differentiation of stem cells from the liver bud into functional hepatocytes for cell therapy.

TLE proteins are a closely related family of vertebrate corepressors. They have no intrinsic DNA binding ability, but are recruited as transcriptional repressors by other sequence specific proteins. TLE proteins and their homologues in other species have been implicated in many developmental processes including neurogenesis, haematopoiesis and the formation of major organs. They have also been implicated in early lineage specification in vertebrates but a direct role in this has not been found in mammals. The aim of my PhD is therefore to analyse the function of TLE proteins in early lineage specification and cell fate decisions using mouse embryonic stem cells (ESCs) as a model. The investigation of this has previously been complicated, firstly by the large array of transcription factors that TLEs interact with and secondly by redundancy between similar TLE proteins hindering loss of function approaches. To circumvent these problems, I have used two complementary experimental strategies. The first was identification of point mutations in TLE1 that affect specific classes of DNA binding. Two of these mutations L743F and R534A were of particular interest and were reversibly overexpressed in ES cells to correlate phenotypes to biochemical activity. The second strategy was the mutation of the two primary TLC genes in ES cells and early mouse embryos, TLE3 and TLE4. Complementary evidence from these approaches revealed a role for TLEs in the promotion of ES cell differentiation by repression of pluripotency/self-renewal associated genes. Additionally, neural specification was increased by TLE1 expression especially by the TLE1 point mutations, highlighting opposing roles for negative effects on mesendodermal differentiation. Early mesoderm/primitive streak was increased by loss of TLE, probably through Wnt antagonism. Anterior endoderm was increased by reduced TLE, but a critical level of TLE was still necessary and TLE1 overexpression also upregulated some anterior endoderm markers suggesting both negative and positive roles for TLE proteins in this process.

During normal cell development, many genes are activated and repressed, usually through epigenetic mechanisms. These are modifications of the DNA and protein within the nucleus that result in changes in gene expression without alteration in DNA sequence. Key proteins for epigenetic modifications are the histone proteins bound to DNA in the nucleus. The best-characterised epigenetic complexes that modify histone proteins are the polycomb group proteins (PcG), comprising polycomb repressive complexes 1 (PRC1) and 2 (PRC2). The repressive modifications generated by these complexes can be removed, and the blocked genes reactivated, by enzymes that are the subject of this project. PRC1 repressive marks are removed by deubiquitinases USP12, USP16 and BAP1, whereas PRC2 marks are removed by demethylases KDM6A, KDM6B and potentially UTY. During the development of cancer, the regulation of many genes becomes abnormal, allowing the cells to escape normal growth restrictions. In this thesis, the expression of this set of chromatin-modifying enzymes in a leukaemia cell line was investigated. The FANTOM consortium has been helping to understand patterns of gene expression for over 10 years. The FANTOM4 dataset described changes in gene expression and promoter usage during differentiation of the THP-1 acute monocytic leukaemia cell line, using CAGE (Cap Analysis of Gene Expression) technology. This human monocyte-like cancer cell line can be stimulated with phorbol esters to halt proliferation and differentiate into macrophages. However, the FANTOM4 time course did not capture detailed mechanisms of regulatory factors in macrophage differentiation due to sparse time points and low read coverage. The main aim of this project was therefore to repeat the time course with tighter time points and deeper sequencing of the transcriptome to develop a very precise picture of sequential activation of gene expression, transcription start site (TSS) usage and the activity of enhancers during the transition from proliferating monocytes to differentiated macrophage phenotype of the THP-1 leukaemia cell line, using CAGE. The focus of this research was on the chromatin-modifying enzymes, but other key cell cycle and macrophage genes have also been examined. The differentiation time course was repeated in triplicate. RNA was extracted and CAGE libraries generated for 18 time points, including the 6 originally studied in FANTOM4. Sequencing results were analysed and normalised using bioinformatics tools. It was shown that analysing 8 samples on one Illumina HiSeq 2500 lane yielded enough read coverage to detect activity from even low expression TSSs, such as those associated with enhancer activity. Clusters of genes which were up- and downregulated at different time points during the differentiation process were identified and characterised. CAGE results for key genes encoding chromatin modifying enzymes and macrophage markers were validated by qRT-PCR. There was a rapid increase of histone demethylase KDM6B mRNA once differentiation was initiated. Histone deubiquitinase USP12 mRNA was also upregulated early in the process. Histone deubiquitinase BAP1 mRNA shows an interesting cyclic regulation pattern which was not seen in the more limited samples of FANTOM4. These interesting chromatin-modifying enzymes and their close paralogues (deubiquitinases USP12, USP16 and BAP1, together with demethylases KDM6A, KDM6B and UTY) were investigated by bioinformatics and genetic tools. USP16 knockout THP-1 cell line was successfully created using CRISPR-Cas9 and its ability to differentiate into macrophages was examined using cell cycle analysis and CAGE sequencing. The USP16 knockout cell line, along with siRNA knock downs of USP12, USP16 and BAP1, was also compared to wildtype THP-1 differentiation using CAGE. Unfortunately, creating other mutant THP-1 cell lines was unsuccessful due to low THP-1 viability after single cell sorting. Investigating KDM6A, KDM6B and UTY using bioinformatics showed that UTY and KDM6A gene expression is positively correlated and this is disrupted in cancer samples. Gene expression and sequence comparison suggested that KDM6A and UTY are coregulated and may act in a similar way in histone demethylation. In summary, the results in this thesis show the transcriptomic changes as the leukaemia cell line ceases proliferation and commences differentiation. Detailed examination suggests that histone modifications are important in the transition between proliferation and differentiation and provide better understanding of regulatory factors in macrophage differentiation and leukaemia.

by Kong Lai Ping, Ada. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 171-189). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.1 --- Development of Hematopoietic Stem Cells and Sites of Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.3 / Chapter 1.2 --- Leukemia and Cell Differentiation --- p.5 / Chapter 1.2.1 --- Leukemia as Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification and Etiology of Leukemia --- p.6 / Chapter 1.2.3 --- Current Modalities for the Treatment of Leukemia --- p.9 / Chapter 1.2.4 --- Leukemia Cell Lines as In Vitro Models for the Study of Myeloid Leukemia --- p.10 / Chapter 1.2.5 --- Cytokines as Inducers of Myeloid Leukemia Cell Differentiation --- p.12 / Chapter 1.2.6 --- The Murine Myeloid Leukemia Cell Line (WEHI- 3B JCS) as an Experimental Cell Model --- p.13 / Chapter 1.3 --- Flavonoids: Properties and Biological Activities --- p.15 / Chapter 1.3.1 --- Chemical Structure and Classification of Flavonoids --- p.15 / Chapter 1.3.2 --- Occurrence and Distribution of Flavonoids --- p.16 / Chapter 1.3.3 --- Biological Properties and Action Mechanisms of Flavonoids --- p.17 / Chapter 1.3.4 --- Effects of Flavonoids on Leukemia --- p.20 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.23 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Cell Lines --- p.26 / Chapter 2.1.2. --- Mice --- p.28 / Chapter 2.1.3 --- Flavonoids --- p.28 / Chapter 2.1.4 --- Recombinant Cytokines --- p.30 / Chapter 2.1.5. --- Physiological Differentiation Inducers ´ؤ Vitamin Analogs --- p.31 / Chapter 2.1.6 --- Monoclonal Antibodies --- p.31 / Chapter 2.1.7 --- "Buffers, Culture Medium and Other Reagents" --- p.33 / Chapter 2.1.8 --- Oligonucleotide Primers and Internal Probes --- p.36 / Chapter 2.1.9 --- Reagents for Cytokine Gene Expression Study --- p.38 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Culture of Tumor Cell Lines --- p.44 / Chapter 2.2.2 --- Determination of Cell Growth and Proliferation --- p.45 / Chapter 2.2.3 --- Colony Assay --- p.46 / Chapter 2.2.4 --- In vivo Tumorigenicity Assay --- p.46 / Chapter 2.2.5 --- Induction of Leukemic Cell Differentiation --- p.47 / Chapter 2.2.6 --- Cell Morphological Study --- p.47 / Chapter 2.2.7 --- Assessment of Differentiation Associated Characteristics --- p.48 / Chapter 2.2.7.1 --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter 2.2.7.2 --- Assay of Plastic Adherence --- p.48 / Chapter 2.2.8 --- Flow Cytometric Analysis --- p.49 / Chapter 2.2.8.1 --- Surface Antigen Immunophenotyping --- p.49 / Chapter 2.2.8.2 --- Assay of Non-specific Esterase Activity --- p.50 / Chapter 2.2.8.3 --- Assay of Phagocytic Activity --- p.50 / Chapter 2.2.8.4 --- Assay of Endocytic Activity --- p.51 / Chapter 2.2.8.5 --- Cell Cycle/DNA Content Evaluation --- p.52 / Chapter 2.2.9 --- Gene Expression Analysis --- p.53 / Chapter 2.2.9.1 --- Cell Lysate Preparation --- p.53 / Chapter 2.2.9.2 --- Total RNA Isolation by cesium chloride isopycnic gradient --- p.53 / Chapter 2.2.9.3 --- Reverse Transcription --- p.54 / Chapter 2.2.9.4 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.2.9.5 --- Agarose Gel Electrophoresis --- p.56 / Chapter 2.2.9.6 --- DIG 3，End Labeling of Oligonucleotide Probes --- p.57 / Chapter 2.2.9.7 --- Dot Blot Hybridization --- p.57 / Chapter 2.2.9.8 --- DIG Chemiluminescent Detection --- p.58 / Chapter 2.2.10 --- DNA Fragmentation Analysis --- p.59 / Chapter 2.2.11 --- Statistical Analysis --- p.60 / Chapter CHAPTER 3: --- EFFECTS OF FLAVONOIDS ON THE PROLIFERATION AND APOPTOSIS OF MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.63 / Chapter 3.2.1 --- Growth-Inhibitory Effects of Flavone on Murine Myeloid Leukemia JCS Cells --- p.63 / Chapter 3.2.2 --- Cytotoxic Effects of Flavone on Murine Lymphocytes and Myeloid Leukemia JCS Cells --- p.67 / Chapter 3.2.3 --- Effects of Different Flavonoids on the Proliferation of Leukemia JCS Cells --- p.70 / Chapter 3.2.4 --- Anti-proliferative Effect of Flavonoids on Different Tumor Cell Lines --- p.74 / Chapter 3.2.5 --- Effects of Flavone and Flavonol on the Cell Cycle Kinetics of JCS Cells --- p.86 / Chapter 3.2.6 --- Induction of DNA Fragmentation of JCS cells by Flavone --- p.89 / Chapter 3.2.7 --- Effect of Flavone on the Clonogenicity of JCS Cells In Vitro and Tumorigenicity In Vivo --- p.92 / Chapter 3.3 --- Discussion --- p.94 / Chapter CHAPTER 4: --- EFFECTS OF FLAVONOIDS ON THE DIFFERENTIATION OF MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Results --- p.100 / Chapter 4.2.1 --- Morphological Changes in Flavonoid-Treated JCS Cells --- p.100 / Chapter 4.2.2 --- Induction of Plastic Adherence in Flavonoid- Treated JCS Cells --- p.106 / Chapter 4.2.3 --- Surface Antigen Immunophenotyping of Differentiating JCS Cells --- p.106 / Chapter 4.2.4 --- NBT-Reducing Activity of Flavonoid-Treated JCS Cells --- p.114 / Chapter 4.2.5 --- Non-specific Esterase Activity of Flavonoid- Treated JCS Cells --- p.115 / Chapter 4.2.6 --- Endocytic Activity of Flavonoid-Treated JCS Cells --- p.116 / Chapter 4.2.7 --- Phagocytic Activity of Flavonoid-Treated JCS Cells --- p.117 / Chapter 4.3 --- Discussion --- p.118 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI- PROLIFERATIVE AND DIFFERENTIAION-INDUCING ACTIVITIES OF FLAVONE ON MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 5.1 --- Introduction --- p.122 / Chapter 5.2 --- Results --- p.125 / Chapter 5.2.1 --- Combinations of Flavone with Physiological Differentiation Inducers on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.1 --- Modulatory Effects of Flavone and All-Trans Retinoic Acid (ATRA) on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.2 --- "Modulatory Effects of Flavone and 1,25- dihydroxyvitamin D3 on the Proliferation and Differentiation of JCS Cells" --- p.130 / Chapter 5.2.2 --- Combinations of Flavone and Cytokines on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.1 --- Modulatory Effects of Flavone and rmlFN-γ on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.2 --- Synergistic Effects of Flavone and rmIL-1 on the Proliferation and Differentiation of JCS Cells --- p.137 / Chapter 5.2.3 --- Modulation of Cytokine Gene Expressionin Flavone-Treated JCS Cells --- p.144 / Chapter 5.3 --- Discussion --- p.159 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.165 / REFERENCES --- p.171

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137

Ratos expostos a antagonistas da angiotensina II no período da lactação apresentam alterações no desenvolvimento renal que persistem na vida adulta promovendo distúrbios progressivos da estrutura e função renal. Esse estudo caracteriza-se por analisar a influência da 1,25-(OH)2-D3 (Vitamina D3) (calcitriol) nas alterações da nefrogênese induzidas pelo bloqueio dos receptores de angiotensina AT1 no período da lactação. Ratos Wistar machos foram divididos em quatro grupos: 1- controle, filhotes de mães que receberam solução de sacarose a 2%; 2- controle+Vit. D, filhotes de mães que receberam solução sacarose a 2% e após o período de lactação (25 dias incluindo o período adaptativo) receberam Vit.D (6ng/dia, calcitriol-abbott); 3- losartan, filhotes de mães que receberam losartan (100 mg/kg/dia) diluído em sacarose 2%; 4- losartan+Vit.D, filhotes de mães que receberam losartan (100 mg/kg/dia) diluído em sacarose 2% e após a lactação (25 dias incluindo o período adaptativo) receberam calcitriol (6ng/dia). O losartan foi diluído em uma solução de sacarose a 2% e administrado em substituição a água (no bebedouro) durante a lactação, enquanto que a Vit. D foi administrada através de mini-osmotic pump (Model 2004 alzet) implantados na região dorsal subctânea 4 dias após o final do período de lactação e mantido durante 28 dias. A pressão arterial sistólica foi aferida 28 dias após a introdução da Vit. D. Em seguida, foram coletadas amostras de sangue e urina das proles para avaliação da função renal e os rins removidos para estudos histológicos, imunoistoquímicos, morfométricos e ELISA. Os ratos expostos ao losartan durante a lactação apresentaram aumento da pressão arterial sistólica (PAS) e alterações de função e estrutura renal caracterizada por aumento do volume urinário, da fração de excreção de sódio e potássio, proteinúria, aumento da área intersticial relativa do córtex renal, atrofia dos túbulos renais, fibrose, inflamação intersticial e diminuição da taxa de filtração glomerular (TFG) e osmolalidade urinária. O tratamento com Vit.D após o período de lactação atenuou as alterações da pressão arterial sistólica, taxa de filtração glomerular, fração de excreção de sódio e potássio, proteinúria, área intersticial relativa e área glomerular relativa, evidenciando efeito protetor nos distúrbios da formação renal e na inflamação. Concluindo, o tratamento com Vit.D introduzido após o término da lactação reduziu as alterações da PAS, da estrutura e função renal induzidas pelo losartan, essas alterações estavam associadas aos distúrbios na diferenciação celular e inflamação que persistiram durante a vida adulta. / Rats exposed to angiotensin II (AII) antagonists during lactation present progressive disturbances in renal development leading to alterations in function and structure that persist and progresses in adult life. This study evaluated the effect of Vitamin D (1,25- (OH) 2-D3) (calcitriol) in the disturbances of renal development provoked by losartan, a AT1 antagonist of AII, exposure during lactation in renal function and structure as well in systolic blood pressure (SBP). Male Wistar rats were divided into four groups: Group 1: control, pups from dams that received 2% sucrose solution; Group 2: control+Vit. D, pups from dams that received sucrose solution 2% and Vit.D (6ng / day) after lactation period (25 days including adaptation period); Group 3: losartan, pups from mothers that received losartan (100 mg / kg / day) diluted in sucrose 2% and Group 4: losartan+Vit.D, pups from mothers who received losartan (100 mg / kg / day) diluted in sucrose 2% and calcitriol (6 ng/ day) after lactation period (25 days including adaptation period). Losartan was administered diluted in drinking water in sucrose solution 2% and Vit. D administered by mini-osmotic pump. Treatment with losartan was conducted during all lactation, while Vit.D was introduced after lactation and maintained during the next 28 days. Then SBP was measured and blood and urine samples were collected of the offspring for the evaluation of renal function and kidney removed for histological, immunohistochemical, morphometric, and ELISA studies. Rats exposed to losartan during lactation showed higher SBP in adulthood life. They also had disturbances in renal function and structure in adulthood. These alterations were characterized by increased urinary volume, fractional sodium and potassium excretion, proteinuria, interstitial area from the renal cortex, renal tubule atrophy, fibrosis, interstitial inflammation and decrease glomerular filtration rate and (GFR) urinary osmolality. In conclusion, treatment with Vit.D after lactation attenuated the increase in SBP and the reduction in sodium and potassium fractional excretion and GFR, of interstitial area. The protector effect of this vitamin D was associated to the reduction of the disturbances in inflammation, cell differentiation and proliferation that persist in adulthood life.