Mecanismos redox estão envolvidos em diversos processos, como sobrevivência, proliferação e diferenciação celular, pela modulação da atividade de quinases, fosfatases e fatores de transcrição, entre outros, através da modificação oxidativa e reversível de resíduos de cisteína. Neste trabalho, nós estudamos processos redox subjacentes a diferenciação osteogênica induzida por BMP2, utilizando linhagens de células MC3T3-E1. Nosso objetivo foi investigar modificações redox como possíveis moduladores do processo de diferenciação osteogênica. Para isso, nós primeiramente caracterizamos a diferenciação osteogênica nas células MC3T3-E1 após o tratamento com BMP2, através da expressão do marcador osteogênico Osteocalcina, da fosforilação do complexo Smad 1/5/8 e da deposição de matriz extracelular calcificada. Análises de expressão gênica por qPCR mostraram que o tratamento com BMP2 resultou no aumento de expressão de NOX4, o que provavelmente leva ao aumento na produção de peróxido de hidrogênio intracelular. Nós investigamos também a modulação de peroxiredoxinas nesse processo e análises de expressão gênica mostraram que não há alterações nos níveis de expressão de Prx1 e 2 durante a diferenciação, mas os ensaios de western blot redox indicam que a Prx1 pode ser oxidada após o tratamento com BMP2, de maneira dose dependente. Outras análises in vitro mostram que células expostas a N-acetilcisteína (NAC) e PEG-catalase apresentam diferenciação osteogênica prejudicada, detectada por baixos níveis de deposição de matriz extracelular calcificada, comparado com células não-tratadas. Além disso, a fosforilação de Smad 1/5/8 são reduzidas nessas condições. Nossos dados sugerem que processos redox podem modular a sinalização celular durante o processo de diferenciação osteogência / Redox mechanisms are involved in several processes, such as cell survival, proliferation and differentiation, among other ways by modulating kinases, phosphatases and transcription factors activity that can occur through reversible and oxidative modification of cysteine residues. We were interested in studying redox processes underlying osteogenic differentiation induced by BMP-2, using MC3T3-E1 cell lineage. Our objective was to investigate redox modifications as possible modulators of the osteogenic differentiation process. We first characterized osteogenic differentiation in MC3T3-E1 cells upon BMP2 treatment, through gene expression of the osteogenic marker Osteocalcin, Smad 1/5/8 (belonging to the BMP-2 pathway) protein phosphorylation and extracellular matrix calcification. Gene expression analysis by qPCR showed that BMP2 treatment resulted in NOX4 upregulation, which probably also leads to hydrogen peroxide production. We have investigated peroxiredoxin modulation in this process, and gene expression analysis shows no significant change in peroxiredoxin 1 and 2 expression levels, but redox western blotting assays indicate that Prx1 can be oxidized after BMP2 treatment, in a dose dependent manner. In vitro analysis shows that cells exposed to N-acetyl-L-cysteine (NAC) and PEG-catalase display impaired osteogenic differentiation, detected by lower levels of calcified extracellular matrix deposition compared with non-treated cells. Moreover, phosphorylation of Smad 1/5/8 complex is reduced under these redox treatments. Our data suggest that redox pathways can modulate cell signaling during the osteogenic differentiation process

The spinal cord is a complex structure responsible for processing sensory inputs and motor outputs. As such, the developmental and spatial organization of cells is highly organized. Diseases affecting the spinal cord, such as Amyotrophic Lateral Sclerosis (ALS), result in the disruption of normal cellular function and intercellular interactions, culminating in neurodegeneration. Deciphering disease mechanisms requires a fundamental understanding of both the normal development of cells within the spinal cord as well as the homeostatic environment that allows for proper function. Biological processes such as cellular differentiation, maturation, and disease progression proceed in an asynchronous and cell type-specific manner. Until recently, bulk measurements of a mixed population of cells have been key in understanding these events. However, bulk measurements can obscure the molecular mechanisms involved in branched or coinciding processes, such as differential transcriptional responses occurring between subpopulations of cells. Measurements in individual cells have largely been restricted to 4 color immunofluorescence assays, which provide a solid but limited view of molecular-level changes. Recently, developments in single cell RNA-sequencing (scRNA-Seq) have provided an avenue of accurately profiling the RNA expression levels of thousands of genes concomitantly in an individual cell. With this increased experimental precision comes the ability to explore pathways that are differentially activated in subpopulations of cells, and to determine the transcriptional programs that underlie complex biological processes. In this dissertation, I will first review the key features of scRNA-Seq and downstream analysis. I will then discuss two applications of scRNA-seq: 1) the in vitro differentiation of mouse embryonic stem cells into motor neurons, and 2) the effect of the ALS-associated gene SOD1G93A expression on cultured motor neurons in a cellular model of ALS.

The lens is an avascular organ that focuses light onto the retina where neural signals are transmitted to the brain and translated into images. Lens transparency is vital for maintaining function. The lens is formed through a transition from organelle-rich epithelial cells to organelle-free fiber cells. Lens cell differentiation, leading to the lack of organelles, provides an environment optimal for minimizing light scatter and maximizing the ability to focus light onto the retina. The process responsible for orchestrating lens cell differentiation has yet to be elucidated. In recent years, data has emerged that led our lab to hypothesize that autophagy is likely involved in lens cell maintenance, cell differentiation, and maintenance of lens transparency. As a first step towards testing this hypothesis, we used RT-PCR, western blot analysis, immunohistochemistry, confocal microscopy, and next generation RNA-Sequencing (RNA-Seq) to examine autophagy genes expressed by the lens to begin mapping their lens function. / by Lyndzie Mattucci. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Key to our understanding of growth regulation in Drosophila would be discovering a ligand that could regulate steroid synthesis. Activins are involved in regulating steroid hormone release in vertebrates. In invertebrates, they most likely function to keep ecdysone levels low to allow the larvae more time to achieve critical weight in order to initiate the metamorphic process. TGF-B(Transforming Growth Factor Beta) is a family of cytokine growth factors. We find that two members of the TGF-B signaling pathway Drosophila Activin (dACT) and Activin-like ligand Dawdle (DAW) signal through the type I receptor Baboon (BABO) and the type II receptor PUNT to primarily activate the transcription factor dSMAD2 and MAD to a lesser extent. One transcription factor brinker (brk) appears to be central to dACT signaling. / In wings dACT signaling is necessary to promote growth however, dACT is not expressed in wings suggesting that dACT is provided through the endocrine system. One possible target tissue of dACT signaling is the ring gland (RG), which synthesizes and secretes the steroid hormone ecdysone (E). Consistent with this idea, using the UAS/GAL-4 system, we find that over-expression of the TGF-B ligand dACT with the neuroendocrine driver 386Y-GAL4 results in an increase in the size of flies. Surprisingly, when we increase the dose with two copies of dACT, it decreases the size of flies also indicating non-autononomous effects. We find that overexpression of the activated form of the dACT type I receptor Baboon (BABO) or brk with the ring gland specific driver phm-GAL4 results in developmental arrest of larvae that stay small and never pupate. The developmental arrest can be overcome by feeding larvae E, suggesting that dACT represses E through brk. These results suggest a model where dACT signaling activates brk which inhibits E. We picked three cytochrome P450 enzymes: phantom (PHM), disembodied (DIS) and spookier (SPKR). / PHM is not regulated by any component in the dACT signaling pathway however, we find DIS and SPKR are down-regulated through brk. MAD and dSmad2 bind to a Smad binding site and MAD out-competes dSMAD2. We find no evidence that Drosophila insulin-like peptides (DILPS)/PI3- Kinase or Ras signal through the dActivin signaling pathway. / by Scott C. Gesualdi. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.

Eukaryotic nuclear DNA is folded with histone and non-histone proteins into chromatin, a nucleoprotein structure regulated by histone post-translational modifications and substitution with histone variants. Chromatin mediates processes such as DNA damage repair, cell differentiation, gene silencing, and centromere specification. Mistaken inheritance of chromatin-mediated gene silencing, for instance, can cause both aberrant development and cancer. Gene silencing at pericentromeres and centromeres, which can be attained through obstruction of transcription as well as through recruitment of specific RNA-degrading proteins, is essential for centromere specification. However, the molecular mechanisms of these processes are not yet thoroughly understood, and therefore they will be the focus of this thesis. A structure termed heterochromatin, for which the essential hallmark is histone H3 lysine 9 methylation (H3K9me), preferentially assembles at repetitive DNA such as pericentric regions, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNA interference (RNAi) machinery is required for heterochromatin assembly over DNA repeats in diverse organisms by targeting histone-modifying activities. Surprisingly, RNA splicing factors are also required for this process. A widely-held model derived from studies in fission yeast is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, we discovered the requirement of four non-essential splicing factors for pericentric heterochromatin assembly, allowing us to more clearly address the role of splicing in heterochromatin assembly. Sequencing total cellular RNA from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors, which correspond to strong reduction in levels of a central RNAi protein, Argonaute. Moreover, introducing cDNA versions of RNAi factors significantly restores pericentric heterochromatin in splicing mutants. We also found that mutation of splicing factors affects telomeric heterochromatin, and replacement of mis-spliced factor tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres. In addition to post-translational modifications, chromatin silencing can be regulated by histone variants such as H2A.Z. The incorporation of H2A.Z into chromatin regulates chromatin structure and gene expression. The Swr1 chromatin remodeling complex deposits H2A.Z in budding yeast and mammals. Here we characterize a novel component of the fission yeast Swr1 complex, Msc1, which is a Jumonji domain protein frequently associated with histone demethylation. We found that Msc1 is required for Swr1-mediated incorporation of H2A.Z into chromatin at gene promoters. We demonstrated that H2A.Z is required for the expression of CENP-C, which in turn regulates centromere silencing and chromosome segregation. Together, these results show that chromatin silencing at pericentromeres and centromeres is mediated by splicing factors and H2A.Z, respectively, to promote proper regulation of other chromatin factors, thus ensuring faithful chromosome segregation.

Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function. This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development. In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis. Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.

Chan Sin-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 154-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Contents --- p.viii / Abbreviations --- p.xiii / List of Figures and Tables --- p.xvi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Ontogeny of hematopoiesis --- p.1 / Chapter 1.1.2 --- Hiercharay of hematopoiesis --- p.2 / Chapter 1.2 --- Regulation of hematopoiesis --- p.5 / Chapter 1.2.1 --- Bone marrow stromal cell --- p.5 / Chapter 1.2.2 --- Hematopoietic growth factor --- p.6 / Chapter 1.2.3 --- Hematopoietic growth factor receptors and signal transduction --- p.10 / Chapter 1.2.4 --- Transcriptional regulation of myeloid cell development --- p.11 / Chapter 1.3 --- Deregulated hematopoiesis - Leukemia --- p.20 / Chapter 1.3.1 --- Classification of leukemia --- p.20 / Chapter 1.3.2 --- Molecular basis of leukemia --- p.20 / Chapter 1.4 --- Prostaglandin E2 induced WEHI-3B JCS and JCS-14 cell differentiation --- p.22 / Chapter 1.4.1 --- Induced leukemia cell differentiation --- p.22 / Chapter 1.4.2 --- Inducer of cell differentiation - Prostaglandin E2 --- p.22 / Chapter 1.4.3 --- WEHI-3B JCS and subline JCS-14 cells --- p.24 / Chapter 1.5 --- The aims of study --- p.26 / Chapter Chapter Two --- Identification of differentially expressed genes during PGE2-induced WEHI-3B JCS-14 cell differentiation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Strategy for studying PGE2-induced JCS-14 cell differentiation --- p.28 / Chapter 2.1.2 --- Method for studying differential gene expression: Microarry Technology --- p.29 / Chapter 2.2 --- Materials --- p.32 / Chapter 2.2.1 --- Cell line --- p.32 / Chapter 2.2.2 --- AtlasT M Mouse cDNA Expression Array --- p.32 / Chapter 2.2.3 --- Chemicals --- p.32 / Chapter 2.2.4 --- Solutions and buffers --- p.33 / Chapter 2.2.5 --- Reagents --- p.34 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Morphological study of PGE2-induced JCS-14 cell differentiation --- p.35 / Chapter 2.3.2 --- Preparation of total RNA from PGE2-induced JCS-14 cells --- p.35 / Chapter 2.3.2.1 --- Preparation of cell lysates --- p.35 / Chapter 2.3.2.2 --- Isolation of total RNA --- p.35 / Chapter 2.3.3 --- Preparation of cDNA probes --- p.36 / Chapter 2.3.3.1 --- Probe synthesis from total RNA --- p.36 / Chapter 2.3.3.2 --- Purification of the labeled cDNA probes --- p.37 / Chapter 2.3.4 --- Hybridization cDNA probes to the Atlas Array and stringency wash --- p.37 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Morphological changes in PGE2-treated JCS-14 cells --- p.39 / Chapter 2.4.2 --- Analysis of total RNA from PGE2-induced JCS-14 cells --- p.43 / Chapter 2.4.3 --- Hybridization of cDNA probes to AtlasT M cDNA Expression Array --- p.45 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Morphological study of JCS-14 cell differentiation --- p.73 / Chapter 2.5.2 --- Differentiation commitment of JCS-14 cell under PGE2 induction --- p.73 / Chapter 2.5.3 --- Gene expression profile by microarray --- p.74 / Chapter 2.5.4 --- Gene expression profile of 5 hours PGE2-induced JCS-14 cells --- p.74 / Chapter 2.5.5 --- Further analysis of regulatory genes in PGE2-induced JCS-14 cell differentiation --- p.77 / Chapter Chapter Three --- Expression profile of identified genes in WEHI-3B JCS-14 and JCS cell differentiation / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.1.1 --- Quantitation of mRNA by Real time RT-PCR --- p.80 / Chapter 3.1.2 --- Relative quantitation of gene expression --- p.83 / Chapter 3.2 --- Materials --- p.85 / Chapter 3.2.1 --- Cell lines --- p.85 / Chapter 3.2.2 --- SYBR® Green PCR core kit --- p.85 / Chapter 3.2.3 --- Chemicals --- p.85 / Chapter 3.2.4 --- Solutions and buffers --- p.86 / Chapter 3.2.5 --- Enzymes and nucleic acids --- p.86 / Chapter 3.3 --- Methods --- p.88 / Chapter 3.3.1 --- Preparation of total RNA from PGE2-induced JCS-14 and JCS cells --- p.88 / Chapter 3.3.1.1 --- Preparation of cell lysates --- p.88 / Chapter 3.3.1.2 --- Isolation of total RNA --- p.88 / Chapter 3.3.2 --- Reverse transcription (RT) --- p.88 / Chapter 3.3.3 --- Design of real-time PCR primers --- p.88 / Chapter 3.3.4 --- Determination of relative efficiency of target and reference amplification by validation experiment --- p.89 / Chapter 3.3.5 --- Confirmation of expression profile of identified genes in JCS-14 and JCS cells by comparative CT method in real-time PCR --- p.90 / Chapter 3.4 --- Results --- p.91 / Chapter 3.4.1 --- Analysis of total RNA from PGE2-induced JCS-14 and JCS cells --- p.91 / Chapter 3.4.2 --- Validation experiment of real-time PCR primers --- p.93 / Chapter 3.4.3 --- Expression profile of specific genes in JCS-14 and JCS cells by comparative CT method --- p.101 / Chapter 3.5 --- Discussion --- p.114 / Chapter 3.5.1 --- Study of gene expression profiles in JCS-14 and JCS cell differentiation --- p.114 / Chapter 3.5.2 --- Transcription analysis by real-time PCR --- p.114 / Chapter 3.5.3 --- Gene expression profiles during PGE2-induced JCS-14 and JCS cell differentiation --- p.115 / Chapter Chapter Four --- Inhibition of specific gene expression in WEHI-3B JCS-14 and JCS cells using antisense blocking technique / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.1.1 --- Antisense technique --- p.122 / Chapter 4.1.2 --- Design of antisense oligonucleotides --- p.125 / Chapter 4.1.3 --- Transfer of oligonucleotides to cells --- p.128 / Chapter 4.2 --- Materials --- p.129 / Chapter 4.2.1 --- Cell lines --- p.129 / Chapter 4.2.2 --- Chemicals --- p.129 / Chapter 4.2.3 --- Reagents --- p.129 / Chapter 4.2.4 --- Solutions --- p.129 / Chapter 4.3 --- Methods --- p.131 / Chapter 4.3.1 --- Design of antisense oligonucleotides --- p.131 / Chapter 4.3.2 --- Transfection of oligonucleotides into cells --- p.134 / Chapter 4.3.3 --- Morphological study of PGE2-induced JCS-14 and JCS cells --- p.134 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- Effect of antisense oligonucleotides on JCS-14 cell differentiation --- p.135 / Chapter 4.4.2 --- Effect of antisense oligonucleotides on JCS cell differentiation --- p.136 / Chapter 4.5 --- Discussion --- p.146 / Chapter 4.5.1 --- Effects of antisense B-myb on JCS-14 and JCS cell differentiation --- p.146 / Chapter 4.5.2 --- Effects of antisense thyroid hormone receptor (c-erbA) and transcription terminator factor (TTF-1) on JCS-14 and JCS cell differentiation --- p.147 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Introduction --- p.148 / Chapter 5.2 --- Differentiation program triggered by Prostaglandin E2 --- p.148 / Chapter 5.2.1 --- Lineage preference during differentiation --- p.148 / Chapter 5.2.2 --- Differentially expressed genes during PGE2-induced JCS-14 cell differentiation --- p.149 / Chapter 5.2.3 --- Expression patterns of the three differentially expressed genes in PGE2-induced JCS-14 and JCS cells --- p.149 / Chapter 5.2.4 --- Antisense blocking during differentiation --- p.151 / Chapter 5.3 --- Further studies --- p.152 / References --- p.154

Yin Xiao-Lu. / "February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Neuroblastoma (NB) é a forma mais indiferenciada de tumores neuroblásticos e a principal causa de morte por câncer pediátrico. Alterações epigenéticas interagem em todas as etapas do desenvolvimento do câncer, promovendo a progressão tumoral. A remodelação da cromatina é influenciada pela acetilação de histonas e a metilação de DNA. Acetiltransferases de histona (HATs), desacetilases de histonas (HDAC) e metiltransferase de DNA (DNMTs) são alvos de estratégias terapêuticas em tumores. Os retinoides agem nas vias de diferenciação celular, anti-proliferação e pró-apoptose. Nesse trabalho, é proposto que a combinação desses moduladores epigenéticos e de diferenciação em linhagens de células de NB humano é mais efetiva que os agentes isolados. Os tratamentos induziram mudanças na expressão de marcadores de diferenciação e indiferenciação, como c-Myc, β-3tubulina, NeuN e Bmi1, e alterações morfológicas nas duas linhagens celulares utilizadas, SK-N-BE(2) e SH-SY5Y. Os dados encontrados podem contribuir para uma melhor compreensão dos mecanismos moleculares dos moduladores retinoides e epigenéticos em NB capazes de acrescentar melhorias nas atuais estratégias terapêuticas. / Neuroblastoma (NB) is the most undifferentiated form of neuroblastic tumors and the leading cause of death from pediatric cancer. Epigenetic changes interact at all stages of cancer development, promoting tumor progression. Chromatin remodeling is influenced by histone acetylation and DNA methylation. Histone acetyltransferases (HATs), histone deacetylases (HDAC), and DNA methyltransferase (DNMTs) are targets for therapeutic strategies in cancer. Retinoids act on cell differentiation pathways and display anti-proliferation and pro-apoptotic actions. In the present research we examined the effects of combining epigenetic modulators and a retinoid receptor agonist in human NB cells. The retinoid all trans-retinoic acid (ATRA) combined with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase was more effective than any drug given alone in impairing the proliferation of SH-SY5Y and SK-N-BE(2) NB cells. In addition, the treatments induced differential changes in the expression of differentiation markers including c-Myc, β-3tubulin, NeuN and Bmi1, and morphological changes in SK-N-BE(2) e SH-SY5Y cell lines. The data contribute to a better understanding of the molecular mechanisms of retinoid modulators and epigenetic in NB able to add improvements in current therapeutic strategies.