Thesis (M.S.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Diss. (sammanfattning Stockholm : Karol. inst. / Härtill 4 uppsatser.

In conclusion, the results suggested the important role of Ncl in driving the optic vesicle formation during early eye development. / The eye is a complex sense organ. It develops from different embryonic origins that including neural ectoderm, surface ectoderm, neural crest and paraxial mesoderm. Morphogenetic waves occur during eye development involve timely interactions of transcription factors and inductive signaling to ensure the correct temporal and spatial development of different components. Genetic studies of congenital eye defects, especially mutation screening and gene targeting, have provided the information about the molecular regulation in the complex processes of eye development. However, our knowledge of the basic genetic pathways that regulate the normal embryonic eye formation is incomplete. / Though the developing eye is believed to be highly specialized extension from the developing neural tube, the formation of major eye structure involves independent coordination of inductive interactions and regional specifications; formation of neural connections between retina and optic tectum; and maturation to a functional eye. There is not much information about eye-specific expression in early embryonic period. In this study, microarray was used to profile the molecular changes occurring in the developing mouse eye between the stage of optic vesicle evagination at E9.5 and completion of basic eye formation at P0. Differentially expressed transcription factor and signaling molecules, including nucleolin gene (Ncl), in the early developing eye were displayed. Temporal expression patterns were confirmed by quantitative real time PCR and spatial expressions patterns were confirmed by the whole-mount in situ hybridization. siRNA and overexpression vector targeting nucleolin transcript was designed to study their roles in the early eye morphogenesis during mouse embryogenesis in vitro. The loss of function phenotype after nucleolin knockdown was demonstrated by the absence of early optic vesicles with normal neural tube in the developing mouse embryos. Ectopic optic vesicle in developing mouse embryo was resulted under overexpression of Ncl . With the aim to study the biological roles of Ncl in mouse embryonic eye development in vivo, both conventional and conditional knockout techniques were attempted. The expression and functional studies revealed that a new neural tube independent signaling pathway regulated in the induction and formation of optic vesicles in the early eye formation. / Tang, Ling Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3294. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 138-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114

Ng, Sze Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-124). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.1.1 --- Pluripotent markers --- p.2 / Chapter 1.1.1.2 --- Germ layers' markers --- p.3 / Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4 / Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4 / Chapter 1.1.2.2 --- Feeder free mESCs --- p.4 / Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5 / Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6 / Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7 / Chapter 1.2.1 --- Background --- p.7 / Chapter 1.2.2 --- Current treatments --- p.8 / Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9 / Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10 / Chapter 1.3 --- Cardiac gene markers --- p.13 / Chapter 1.3.1 --- Atrial-specific --- p.13 / Chapter 1.3.2 --- Ventricular-specific --- p.19 / Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27 / Chapter 1.5 --- Cell cycle in ESCs --- p.29 / Chapter 1.5.1 --- Cell cycle --- p.29 / Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30 / Chapter 1.6 --- Potassium (K+) channels --- p.31 / Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32 / Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32 / Chapter 1.7 --- Objectives and significances --- p.33 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35 / Chapter 2.1.1 --- Derivation of MEF --- p.3 5 / Chapter 2.1.2 --- MEF culture --- p.37 / Chapter 2.1.3 --- Irradiation of MEF --- p.37 / Chapter 2.2 --- mESC culture and their differentiation --- p.38 / Chapter 2.2.1 --- mESC culture --- p.38 / Chapter 2.2.2 --- Differentiation of mESCs --- p.39 / Chapter 2.3 --- Subcloning --- p.40 / Chapter 2.3.1 --- Amplification of Irx4 --- p.40 / Chapter 2.3.2 --- Purification of DNA products --- p.41 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.42 / Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43 / Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44 / Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45 / Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45 / Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45 / Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47 / Chapter 2.4.1 --- Lentivirus packaging --- p.47 / Chapter 2.4.2 --- Lentivirus titering --- p.48 / Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48 / Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49 / Chapter 2.5 --- Selection of stable clone --- p.49 / Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49 / Chapter 2.6 --- Differentiation of cell lines after selection --- p.50 / Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50 / Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55 / Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55 / Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55 / Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56 / Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57 / Chapter 2.9 --- Apoptotic study --- p.58 / Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59 / Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59 / Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61 / Chapter CHAPTER THREE --- RESULTS --- p.62 / Chapter 3.1 --- mESC culture --- p.62 / Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62 / Chapter 3.2 --- Subcloning --- p.63 / Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63 / Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64 / Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66 / Chapter 3.2.4 --- Confirmation of successful ligation --- p.67 / Chapter 3.3 --- Lentivirus packaging --- p.68 / Chapter 3.3.1 --- Transfection --- p.68 / Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69 / Chapter 3.5 --- FACS --- p.70 / Chapter 3.6 --- Irx4 and iduet clone selection --- p.71 / Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74 / Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74 / Chapter 3.8 --- Differentiation of cell lines after selection --- p.77 / Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77 / Chapter 3.9 --- Gene expression study by qPCR --- p.79 / Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84 / Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86 / Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87 / Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89 / Chapter 3.11.1 --- Apoptotic study --- p.89 / Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91 / Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92 / Chapter CHAPTER FOUR --- DISCUSSION --- p.95 / Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95 / Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99 / Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99 / Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102 / Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105 / Conclusions --- p.108 / References --- p.110

Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Poon, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-124). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.6 / Chapter Chapter 2 --- Literature Reviews --- p.8 / Chapter 2.1 --- Cancer genetics --- p.8 / Chapter 2.1.1 --- Oncogenes and TSGs --- p.8 / Chapter 2.1.2 --- Kundsońةs two-hit event of cancer gene --- p.9 / Chapter 2.2 --- Cancer Epigenetics --- p.9 / Chapter 2.2.1 --- Types of Epigenetic regulation --- p.10 / Chapter 2.2.2 --- DNA methylation in TSGs --- p.10 / Chapter 2.2.2.1 --- Promoter CpG island in DNA methylation --- p.10 / Chapter 2.2.2.2 --- Protection system in DNA methylation --- p.11 / Chapter 2.2.2.3 --- Transcriptional silencing by DNA methylation --- p.11 / Chapter 2.2.2.4 --- DNA methylation of TSG silencing in cancers --- p.13 / Chapter 2.2.3 --- Hypomethylation of the cancer genome --- p.14 / Chapter 2.2.4 --- Clinical relevance of cancer epigenetic --- p.14 / Chapter 2.3 --- EGFR/STAT/SOCS pathway --- p.15 / Chapter 2.3.1 --- General Introduction of the EGFR pathway --- p.15 / Chapter 2.3.2 --- EGFR survival signaling pathways --- p.16 / Chapter 2.3.3 --- EGFR/STAT/SOCS signaling --- p.17 / Chapter 2.3.4 --- EGFR/STAT/SOCS signaling and cancers --- p.18 / Chapter 2.3.4.1 --- EGF and cancers --- p.18 / Chapter 2.3.4.2 --- EGFR/STAT/SOCS pathway and cancers --- p.18 / Chapter 2.3.4.3 --- EGF survival signaling as a target for cancer therapy --- p.19 / Chapter 2.4 --- TSGs in the EGFR/STAT/SOCS pathway --- p.20 / Chapter 2.4.1 --- Suppressors of cytokine signaling (SOCS) family --- p.20 / Chapter 2.4.2 --- Signal transducers and activators of transcription (STATs) family --- p.22 / Chapter 2.4.3 --- Sprouty (SPRY) family --- p.23 / Chapter 2.4.4 --- Protein Inhibitor of Activated STAT (PIASs) family --- p.25 / Chapter 2.4.5 --- Ras and Rab Interactor (RIN) family --- p.26 / Chapter 2.4.6 --- Ras-association domain family (RASSF) --- p.26 / Chapter 2.4.7 --- Glycine N-methyltransferase (GNMT) --- p.28 / Chapter 2.5 --- Nasopharyngeal carcinoma (NPC) --- p.30 / Chapter 2.5.1 --- Epidemiology of NPC --- p.30 / Chapter 2.5.2 --- Histopathology of NPC --- p.30 / Chapter 2.5.3 --- Genetic and epigenetic alteration in NPC --- p.31 / Chapter 2.5.4 --- EGFR signaling in NPC --- p.32 / Chapter 2.6 --- Esophageal squamous cell carcinoma (ESCC) --- p.33 / Chapter 2.6.1 --- Epidemiology of ESCC --- p.34 / Chapter 2.6.2 --- Histopathology of ESCC --- p.34 / Chapter 2.6.3 --- Genetic and epigenetic alteration in ESCC --- p.35 / Chapter 2.6.4 --- EGFR signaling in ESCC --- p.36 / Chapter Chapter 3 --- Materials and Methods --- p.38 / Chapter 3.1 --- General Materials --- p.38 / Chapter 3.1.1 --- "Cell lines, tumor and normal tissue samples" --- p.38 / Chapter 3.1.2 --- Maintenance of cell lines --- p.38 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.39 / Chapter 3.1.4 --- Total RNA extraction --- p.39 / Chapter 3.1.5 --- Genomic DNA extraction --- p.40 / Chapter 3.2 --- General techniques --- p.40 / Chapter 3.2.1 --- Agarose gel electrophoresis of DNA --- p.40 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.40 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.41 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.41 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.41 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.42 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.42 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.42 / Chapter 3.3 --- Preparation of reagents and medium --- p.43 / Chapter 3.4 --- Semi-quatitative Reverse-Transcription (RT) PCR expression analysis --- p.44 / Chapter 3.4.1 --- Reverse transcriptin reaction --- p.44 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.44 / Chapter 3.4.2.1 --- Primers design --- p.44 / Chapter 3.4.2.2 --- PCR reaction --- p.46 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.47 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.47 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.48 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.48 / Chapter 3.5.2.2 --- Primers design --- p.48 / Chapter 3.5.2.3 --- PCR reaction --- p.49 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.50 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.51 / Chapter 3.6.1 --- Sub-cloning of expression vector of candidate genes --- p.51 / Chapter 3.6.1.1 --- Mouse Socsl expression vector --- p.51 / Chapter 3.6.1.2 --- SPRY1 expression vector --- p.51 / Chapter 3.6.1.3 --- GNMT expression vector --- p.52 / Chapter 3.6.2 --- Restriction digestion of cloning vectors and expression --- p.52 / Chapter 3.6.3 --- Ligation of cloning fragments --- p.53 / Chapter 3.6.4 --- Colony formation assay on monolayer culture --- p.53 / Chapter 3.6.5 --- Statistical analysis --- p.54 / Chapter Chapter 4 --- Screening of candidate TSGs in EGFR pathway --- p.55 / Chapter 5.3.3 --- Restoration of GNMT expression by pharmacological demethylation --- p.89 / Chapter 5.3.4 --- Confirmation of the methylation status of GNMT promoter by BGS --- p.90 / Chapter 5.3.5 --- Methylation status of GNMT in ESCC and NPC primary tumors --- p.90 / Chapter 5.3.6 --- GNMT inhibited the growth of tumor cells in-vitro --- p.90 / Chapter 5.3.7 --- Discussion --- p.95 / Chapter Chapter 6 --- General Discussion --- p.100 / Chapter Chapter 7 --- Summary --- p.105 / Chapter Chapter 8 --- Future Study --- p.107 / Reference --- p.109

Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.