The ubiquitin ligase Highwire is responsible for cell-autonomously promoting synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects in synaptic function and extra branching at the axon terminal, corresponding to transient branching that occur in the course of giant synapse formation during metamorphosis. The MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner, suggesting different molecular mechanisms downstream of the MAP kinase pathway govern function and pruning. A novel role for Highwire is revealed, regulating the giant fiber axon’s ability to respond to external cues regulated by Fos. When expression of the transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Obesity is associated with elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), contributing to systemic insulin resistance. Fibroblast growth factor 21 (FGF21) is a vital metabolic and inflammatory regulator, however circulating FGF21 concentrations are elevated in obese individuals. Acute aerobic exercise increases systemic FGF21 in normal-weight individuals, however the effect of acute aerobic exercise on plasma FGF21 response and the relationships with inflammation (IL-6 and TNF-α), insulin resistance, and energy expenditure in obese individuals is unknown. Following 30 minutes of treadmill running at 75% VO2max, plasma FGF21 response, as indicated by area-under-the-curve “with respect to increase” (AUCi) analyses, was attenuated in 12 obese compared to 12 normalweight subjects. Additionally, FGF21 AUCi positively correlated with glucose AUCi, total relative energy expenditure, and relative VO2max, suggesting that cardiorespiratory fitness levels may predict FGF21 response, contributing to the enhanced regulation of glucose and energy metabolism. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

O titânio comercialmente puro (Ti-Cp) e suas ligas destacam-se como biomateriais metálicos devido a sua biocompatibilidade sendo amplamente utilizados. Buscando aprimorar o sucesso clínico dos implantes de Ti-Cp em longo prazo, é necessário revestir a sua superfície, proporcionando uma eficiente ancoragem mecânica do implante com o tecido ósseo. Dentre os diversos revestimentos superficiais destacam-se os revestimentos híbridos orgânicos-inorgânicos à base de precursores alcóxidos de silício, obtidos via processo sol-gel. Para que ocorra uma satisfatória adesão do revestimento no substrato, precisa-se levar em consideração a natureza e a preparação da superfície metálica antes da aplicação deste revestimento. Na etapa inicial do trabalho, pré-tratamentos superficiais foram propostos previamente à aplicação dos revestimentos buscando aumentar a aderência entre o titânio e este revestimento híbrido. Utilizou-se três diferentes pré-tratamentos na superfície do Ti-Cp: o "piranha" (ácido sulfúrico e peróxido de hidrogênio), o "kroll" (ácido fluorídrico, ácido nítrico e água) e o "hidróxido de sódio". Em sequência, aplicou-se por processo de dip-coating sobre as superfícies tratadas, um revestimento híbrido produzido a partir dos precursores alcóxidos de silício tetraetoxisilano (TEOS) e metiltrietoxisilano (MTES), obtido pelo processo de sol-gel. Em uma segunda etapa, sobre a superfície do Ti-Cp com o pré-tratamento superficial que proporcionou uma maior aderência do revestimento híbrido ao titânio, aplicou-se por dip-coating, um revestimento híbrido à base de precursores alcóxidos de silício TEOS e MTES com adição de partículas de hidroxiapatita buscando aprimorar a diferenciação celular sobre o revestimento híbrido. O pré-tratamento com o hidróxido de sódio promove o melhor resultado dentre os pré-tratamentos, pois o revestimento híbrido aplicado posteriormente apresenta recobrimento regular e adesão ao substrato de Ti-Cp. Os resultados morfológicos por MEV-FEG mostraram um revestimento híbrido com boa dispersão da hidroxiapatita e um recobrimento regular e adesão ao substrato de Ti-Cp. Nos resultados biológicos Ti-Cp revestido com TEOS/MTES com a presença de partículas de hidroxiapatita obteve uma adesão celular semelhante ao Ti-Cp sem tratamento. Porém este mesmo revestimento não propiciou a proliferação e diferenciação celular. Os resultados indicaram que a combinação de fatores como a sua superfície hidrofóbica (91°) e a presença da hidroxiapatita no revestimento tornou a superfície desorganizada, acarretando em uma superfície com comportamento desfavorável para o desenvolvimento das células-tronco mesenquimais. / Commercially pure titanium (cp-Ti) and its alloys stand out among the metallic materials due to their biocompatibility being widely used in biomaterials. In order to improve the clinical success of cp-Ti implants in the long term, it is necessary to coat the surface, providing an efficient mechanical anchoring of the implant with the bone tissue. Among the various surface coatings are the hybrid coatings based on silicon alkoxide precursors, obtained by the sol-gel process, however, considering the nature and the preparation of the metal surface prior to the application of this coating. In the initial stage of the work, surface pre-treatments were proposed prior to the application of the coatings seeking to increase the adhesion between the titanium and this hybrid coating. Three different pretreatments were used on the cp-Ti surface: "piranha" (sulfuric acid and hydrogen peroxide), "kroll "(hydrofluoric acid, nitric acid and water) and "sodium hydroxide". Subsequently, a hybrid coating produced by the tetraethoxysilane silicon (TEOS) and methyltriethoxysilane (MTES) precursors obtained by the sol-gel process was applied by the dip-coating process onto the treated surfaces. In a second step, on the surface of the cp-Ti with the surface pretreatment that gave a greater adhesion of the hybrid coating to the titanium, dip-coating, a hybrid coating based on precursors silicon alkoxides TEOS and MTES with the addition of hydroxyapatite particles to enhance cell differentiation on the hybrid coating. Pretreatment with sodium hydroxide promotes the best result among the pre-treatments, since the hybrid coating applied afterwards presents regular coating and adhesion to the cp-Ti substrate. The morphological results by SEM-FEG showed a hybrid coating with good dispersion of the hydroxyapatite and a regular coating and adhesion to the cp-Ti substrate. In the biological results Ti-Cp coated with TEOS / MTES with the presence of hydroxyapatite particles obtained a cell adhesion similar to Ti-Cp without treatment. However, this same coating did not promote cell proliferation and differentiation. The results indicated that the combination of factors such as its hydrophobic surface (91°) and the presence of the hydroxyapatite encapsulated in the coating rendering the surface disorganized led to a surface with unfavorable behavior for the development of mesenchymal stem cells.

O objetivo deste trabalho foi avaliar a influência das características química e de rugosidade da superfície de titânio sobre a adesão, proliferação e diferenciação de células semelhantes aos osteoblastos de rato (Osteo-1), cultivados em meio de cultura adicionado de BMP-7. MATERIAL E MÉTODO: Células Osteo-1 foram cultivadas sobre discos de titânio com superfícies:1) lisa, 2) jateada por areia de grânulos grandes e atacada por ácido (SLA) e 3) rugosa SLA e quimicamente modificada e hidrofílica (SLAactive) na presença ou ausência de 20ng/ml de rhBMP- 7 no meio de cultura. A adesão e viabilidade das células Osteo-1 foram analisadas após 24 horas de contato com as superfícies em estudo. A diferenciação celular foi avaliada através da análise do conteúdo de proteína total (PT), conteúdo de colágeno, atividade de fosfatase alcalina (ALPase), em 7, 14 e 21 dias, e da formação de matriz mineralizada, em 21 dias. Os resultados foram comparados pela análise de variância (ANOVA) e teste de Tukey. RESULTADOS: A adesão (p=0.3485) e a viabilidade (p=0.5516) celular, o conteúdo de colágeno (p=0.1165) e a formação de matriz mineralizada (p=0.5319) não foram afetados pelas diferentes superfícies ou pela adição de rhBMP-7 ao meio. Células Osteo-1 cultivadas sobre superfície SLA apresentaram um aumento significativo no conteúdo de proteína total aos 21 dias. A relação atividade de ALPase/PT (p=0.0000) foi afetada pelos tratamento e tempo. CONCLUSÃO: Os resultados sugerem que a adição de rhBMP- 7 ao meio de cultura não promoveu efeito sobre a adesão, proliferação e diferenciação de células semelhantes a osteoblastos nas diferentes superfícies testadas. Todas as superfícies de titânio testadas permitiram uma completa expressão do fenótipo de osteoblasto como a mineralização da matriz pela célula Osteo-1. / The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the attachment, proliferation and differentiation of osteoblast-like cells cultured in medium supplemented with bone morphogenetic protein-7 (BMP-7). METHODS: Osteo-1 cells were grown on titanium discs presenting the following surfaces: 1) machined surface, 2) coarse gritblasted and acid-etched (SLA), and 3) modified SLA (SLAactive) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The attachment and viability of osteo-1 cells were evaluated after 24 h. Cell differentiation was evaluated by analysis of total protein content (TP), collagen content and alkaline phosphatase (ALPase) activity at 7, 14 and 21 days and of mineralized matrix formation at 21 days. The results were compared by analysis of variance (ANOVA) and Tukey\'s test. RESULTS: Cell attachment (p=0.3485), cell viability (p=0.5516), collagen content (p=0.1165) and mineralized matrix formation (p=0.5319) were not affected by the different surfaces or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surface presented a significant increase in TP at 21 days. The ALPase/TP ratio (p=0.0000) was affected by treatment and time. CONCLUSION: The results suggest that the addition of rhBMP-7 to the culture medium did not promote any effect on the adhesion, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed permitted the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

Dai, Zhuojun. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references. / Abstracts also in Chinese. / Title from PDF title page (viewed on 15, September, 2016).

by Chan Yick Bun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 145-165). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / List of Figures --- p.XVI / List of Tables --- p.XVII / Chapter Chapter One --- General introduction / Chapter 1.1 --- Leukemia: an overview --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Classification of leukemia --- p.1 / Chapter 1.1.3 --- Origin of leukemia --- p.3 / Chapter 1.1.4 --- Treatment of leukemia --- p.5 / Chapter 1.2 --- Introduction of leukemia cell re-differentiation --- p.8 / Chapter 1.2.1 --- Introduction --- p.8 / Chapter 1.2.2 --- Inducers of cell differentiation --- p.8 / Chapter 1.2.3 --- Genes involved in myeloid leukemia cell differentiation --- p.11 / Chapter 1.2.3.1 --- Transcription factors --- p.11 / Chapter 1.2.3.2 --- Signal transduction cascades --- p.16 / Chapter 1.2.3.3 --- Receptors --- p.18 / Chapter 1.2.3.4 --- Cytokines --- p.19 / Chapter 1.3 --- Tumor necrosis factor alpha induced WEHI 3B JCS cell differentiation --- p.21 / Chapter 1.3.1 --- Introduction --- p.21 / Chapter 1.3.2 --- Tumor necrosis factor alpha --- p.21 / Chapter 1.3.3 --- WEHI 3B JCS cells --- p.23 / Chapter 1.4 --- Aims of study --- p.25 / Chapter Chapter Two --- Isolation of differentially expressed genes during TNF-α induced WEHI 3B JCS cell differentiation / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.1.1 --- Overview of differential genes screening methods --- p.26 / Chapter 2.1.2 --- Differential hybridization for analysis of gene expression profiles --- p.29 / Chapter 2.1.3 --- Factors affect differential hybridization --- p.33 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.2.1 --- Cell line --- p.35 / Chapter 2.2.2 --- Mouse brain cDNA library --- p.35 / Chapter 2.2.3 --- E.coli strains --- p.35 / Chapter 2.2.3 --- Kits --- p.35 / Chapter 2.2.5 --- Chemicals --- p.35 / Chapter 2.2.6 --- Solutions and buffers --- p.36 / Chapter 2.2.7 --- Enzymes and reagents --- p.37 / Chapter 2.3 --- Methods --- p.38 / Chapter 2.3.1 --- Preparation of total RNA from TNF-a induced WEHI 3B JCS cells --- p.38 / Chapter 2.3.1.1 --- Preparation of cell lysates --- p.38 / Chapter 2.3.1.2 --- Extraction of total RNA --- p.38 / Chapter 2.3.2 --- Preparation of cDNA clones from cDNA library --- p.39 / Chapter 2.3.2.1 --- Rescue of phagemids from cDNA library --- p.39 / Chapter 2.3.2.2 --- Preparation of plasmids --- p.39 / Chapter 2.3.3 --- Primary differential hybridization --- p.40 / Chapter 2.3.3.1 --- Preparation of cDNA blots --- p.40 / Chapter 2.3.3.2 --- Preparation of cDNA probes --- p.40 / Chapter 2.3.3.3 --- Primary differential hybridization --- p.41 / Chapter 2.3.4 --- Subcloning of putative differential cDNA clones --- p.42 / Chapter 2.3.4.1 --- Preparation of DH5a competent cells --- p.42 / Chapter 2.3.4.2 --- Transformation of cDNA clones --- p.42 / Chapter 2.3.5 --- Secondary differential hybridization --- p.42 / Chapter 2.3.5.1 --- Preparation ofcDNA blots --- p.42 / Chapter 2.3.5.2 --- Secondary differential hybridization --- p.43 / Chapter 2.4 --- Results --- p.44 / Chapter 2.4.1 --- Analysis of total RNA prepared from TNF-α induced WEHI 3B JCS cells --- p.44 / Chapter 2.4.2 --- Spectrophotometric analysis of plasmid DNA --- p.46 / Chapter 2.4.3 --- Primary differential hybridization --- p.48 / Chapter 2.4.4 --- Secondary differential hybridization --- p.58 / Chapter 2.4.5 --- Comparison of two rounds of differential hybridization --- p.61 / Chapter 2.5 --- Discussions --- p.63 / Chapter 2.5.1 --- Study of gene expression profile by differential hybridization --- p.63 / Chapter 2.5.1.1 --- cDNA library --- p.63 / Chapter 2.5.1.2 --- Blots --- p.64 / Chapter 2.5.2 --- Two rounds of differential hybridization --- p.66 / Chapter 2.5.3 --- Comparison of two rounds of differential hybridization --- p.68 / Chapter Chapter Three --- Sequence analysis of putative differentially expressed genes / Chapter 3.1 --- Introduction --- p.70 / Chapter 3.1.1 --- Basic structure of cDNA clones --- p.70 / Chapter 3.1.2 --- Strategies for DNA sequencing --- p.71 / Chapter 3.1.2.1 --- Primer walking --- p.71 / Chapter 3.1.2.2 --- Restriction digestion and subcloning --- p.71 / Chapter 3.1.2.3 --- Nested deletion sets --- p.72 / Chapter 3.1.2.4 --- Shotgun sequencing --- p.72 / Chapter 3.1.2.5 --- Other sequencing strategies --- p.73 / Chapter 3.1.3 --- Sequence alignment and database search --- p.74 / Chapter 3.1.3.1 --- Sequence database --- p.74 / Chapter 3.1.3.2 --- Sequence alignment --- p.74 / Chapter 3.1.3.3 --- BLAST algorithm --- p.75 / Chapter 3.2 --- Materials --- p.76 / Chapter 3.2.1 --- Kits --- p.76 / Chapter 3.2.2 --- Restriction enzymes --- p.76 / Chapter 3.2.3 --- Solutions and buffers --- p.76 / Chapter 3.2.4 --- Enzymes and reagents --- p.77 / Chapter 3.3 --- Methods --- p.78 / Chapter 3.3.1 --- Restriction digestion --- p.78 / Chapter 3.3.2 --- Subcloning --- p.79 / Chapter 3.3.2.1 --- Gel purification --- p.79 / Chapter 3.3.2.2 --- Ligation --- p.79 / Chapter 3.3.2.3 --- Transformation --- p.80 / Chapter 3.3.3 --- Shotgun sequencing --- p.80 / Chapter 3.3.4 --- Sequencing reaction --- p.81 / Chapter 3.3.4.1 --- Preparation of sequencing gel --- p.81 / Chapter 3.3.4.2 --- Sequencing reaction --- p.81 / Chapter 3.4 --- Results --- p.83 / Chapter 3.4.1 --- Restriction mapping of cDNA inserts --- p.83 / Chapter 3.4.2 --- Sequencing results --- p.85 / Chapter 3.4.3 --- Sequence analysis --- p.90 / Chapter 3.5 --- Discussions --- p.103 / Chapter 3.5.1 --- Sequencing strategies --- p.103 / Chapter 3.5.2 --- Sequence analysis --- p.104 / Chapter Chapter Four --- Characterization of the putative differentially expressed genes / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Midazolam induced WEHI 3B JCS cells differentiation --- p.107 / Chapter 4.1.2 --- Gene expression profiles in embryogenesis --- p.108 / Chapter 4.2 --- Materials --- p.110 / Chapter 4.2.1 --- Mouse embryo multiple tissue Northern (MTN´ёØ) blot --- p.110 / Chapter 4.2.2 --- Megaprime´ёØ DNA labelling system --- p.110 / Chapter 4.2.3 --- Chemicals --- p.110 / Chapter 4.2.3 --- Solutions and buffers --- p.111 / Chapter 4.3 --- Methods --- p.112 / Chapter 4.3.1 --- Preparation of Northern blots --- p.112 / Chapter 4.3.1.1 --- Preparation of total RNA from midazolam induced WEHI 3B JCS cells --- p.112 / Chapter 4.3.1.2 --- Preparation of Northern blots --- p.112 / Chapter 4.3.2 --- Preparation of DNA probes --- p.113 / Chapter 4.3.2.1 --- Preparation of DNA templates --- p.113 / Chapter 4.3.2.2 --- Preparation of 32P labelled probes --- p.114 / Chapter 4.3.3 --- Northern blot analysis --- p.115 / Chapter 4.3.3.1 --- Northern hybridization --- p.115 / Chapter 4.3.3.2 --- Stripping of Northern blot --- p.115 / Chapter 4.4 --- Results --- p.117 / Chapter 4.4.1 --- Analysis of midazolam induced JCS cells total RNA --- p.117 / Chapter 4.4.2 --- Preparation of DNA templates for probe syntheses --- p.119 / Chapter 4.4.3 --- Northern Hybridization --- p.121 / Chapter 4.4.4 --- Comparison of the results of differential hybridization and Northern hybridization --- p.126 / Chapter 4.5 --- Discussions --- p.127 / Chapter 4.5.1 --- Northern hybridization --- p.127 / Chapter 4.5.1.1 --- Gene expression patterns under TNF-α induction --- p.127 / Chapter 4.5.1.2 --- Normalization of Northern hybridization --- p.129 / Chapter 4.5.1.3 --- Gene expression patterns under midazolam induction --- p.130 / Chapter 4.5.1.4 --- Gene expression pattern during embryo development --- p.133 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Identification of differentially expressed genes in TNF-α induced WEHI 3B JCS diffentiation --- p.135 / Chapter 5.2 --- Differentially expressed genes and myeloid leukemia cell differentiation --- p.137 / Chapter 5.3 --- Differentially expressed genes and embryogenesis --- p.142 / Chapter 5.4 --- Further studies --- p.144 / References --- p.145

by Yip Mei Chu Pandora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 207-233). / Abstract also in Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER ONE ... --- GENERAL INTRODUCTION / Chapter 1.1 --- the blood cells formation - hematopoiesis --- p.1 / Chapter 1.1.1 --- Hierarchy of hematopoiesis --- p.2 / Chapter 1.1.2 --- Malfunction in the process of hematopoiesis - hematologic neoplasia - Leukemia --- p.6 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Differentiation therapy ´ؤ a new hope in the treatment of leukemia --- p.9 / Chapter 1.2 --- Understanding the pathogenesis of leukemia --- p.12 / Chapter 1.2.1 --- General regulation of hematopoiesis --- p.12 / Chapter 1.2.2 --- Regulation of the differentiation of myeloid lineage --- p.15 / Chapter 1.2.2.1 --- Regulation of myeloid cell differentiation by hematopoietic regulatory protein --- p.16 / Chapter 1.2.2.2 --- Signal transduction pathways in myeloid cell differentiation --- p.20 / Chapter 1.2.2.3 --- Gene regulation of myeloid cell differentiation --- p.22 / Chapter 1.2.2.3.1 --- Transcription factors --- p.23 / Chapter 1.2.2.3.2 --- Myeloid specific genes --- p.31 / Chapter 1.2.2.3.3 --- Protooncogenes and tumor suppressor genes --- p.37 / Chapter 1.2.2.3.4 --- Homeobox genes --- p.42 / Chapter 1.2.2.3.5 --- Cell cycle control in myeloid growth and differentiation --- p.47 / Chapter 1.3 --- Induction of differentiation in myeloid leukemia cell --- p.48 / Chapter 1.3.1 --- Induced myeloid leukemia cell differentiation --- p.48 / Chapter 1.3.2 --- Inducers of myeloid cell differentiation --- p.52 / Chapter 1.3.3 --- Chemical inducers ´ؤ Flavonoids --- p.57 / Chapter 1.3.4 --- Murine myeloid leukemia cell ´ؤ WEHI-3B JCS --- p.60 / Chapter 1.4 --- Aim of study --- p.53 / Chapter CHAPTER TWO ... --- ISOLATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED DURING BIOCHANIN A INDUCED WEHI-3B (JCS) MYELOID LEUKEMIA CELL DIFFERENTIATION / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.1.1 --- Strategy for searching differentially expressed genes - RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP- PCR) --- p.65 / Chapter 2.1.2 --- Reamplification of PCR products by Touchdown PCR --- p.67 / Chapter 2.1.3 --- Methods for eliminating false positives : Dot blot hybridization screening --- p.68 / Chapter 2.2 --- Materials --- p.70 / Chapter 2.2.1 --- "Cell line, Bacterial strain and Vector" --- p.70 / Chapter 2.2.2 --- Chemicals --- p.70 / Chapter 2.2.3 --- Reagents and nucleic acids --- p.71 / Chapter 2.2.4 --- Kits --- p.72 / Chapter 2.2.5 --- Solutions --- p.72 / Chapter 2.2.6 --- Equipments --- p.73 / Chapter 2.3 --- Methods --- p.74 / Chapter 2.3.1 --- Induction of murine myeloid leukemia cell line -WEHI-3B (JCS) cells by biochanin-A --- p.74 / Chapter 2.3.2 --- Isolation of total RNA by guanidium thiocyanate cesium chloride ultracentrifugation --- p.74 / Chapter 2.3.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.75 / Chapter 2.3.3.1 --- Synthesis of first strand cDNA --- p.75 / Chapter 2.3.3.2 --- Normalization of RNA samples --- p.75 / Chapter 2.3.3.3 --- RAP-PCR --- p.76 / Chapter 2.3.3.4 --- Reamplification of differentially amplified fragment --- p.77 / Chapter 2.3.4 --- First round dot blot hybridization screening --- p.78 / Chapter 2.3.4.1 --- Dot blot --- p.78 / Chapter 2.3.4.2 --- Preparation of cDNA probe --- p.79 / Chapter 2.3.4.3 --- 32P-labelling of cDNA probe --- p.79 / Chapter 2.3.4.4 --- Removal of unincorporated probe by NICK´ёØ column --- p.80 / Chapter 2.3.4.5 --- Estimation of 32P labelling efficiency by scintillation counting --- p.80 / Chapter 2.3.4.6 --- Prehybridization and hybridization --- p.81 / Chapter 2.3.4.7 --- Quantitation of hybridization signal by scanning densitometry --- p.81 / Chapter 2.3.5 --- Second round dot blot hybridization screening --- p.81 / Chapter 2.3.5.1 --- Subcloning of differentially amplified fragments --- p.82 / Chapter 2.3.5.1.1 --- Preparation of vector DNA --- p.82 / Chapter 2.3.5.1.2 --- Synthesis of blunt end PCR product --- p.84 / Chapter 2.3.5.1.3 --- Blunt end ligation --- p.34 / Chapter 2.3.5.1.4 --- Transformation --- p.85 / Chapter 2.3.5.1.5 --- Selection and confirmation by polymerase chain reaction --- p.85 / Chapter 2.3.5.2 --- Dot blot hybridization screening --- p.85 / Chapter 2.4 --- Results --- p.87 / Chapter 2.4.1 --- Spectrophotometric analysis of total RNA --- p.87 / Chapter 2.4.2 --- Normalization of RNA samples --- p.88 / Chapter 2.4.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.39 / Chapter 2.4.4 --- Reamplification of isolated RAP-PCR products --- p.91 / Chapter 2.4.5 --- First round of dot blot hybridization screening --- p.92 / Chapter 2.4.6 --- Subcloning of differentially amplified fragments --- p.100 / Chapter 2.4.7 --- Second round of dot blot hybridization screening --- p.102 / Chapter 2.4.8 --- Comparison of the first and second round of dot blot hybridization screening --- p.106 / Chapter 2.5 --- Discussion --- p.108 / Chapter 2.5.1 --- RNA fingerprinting by arbitrarily primed PCR --- p.108 / Chapter 2.5.2 --- Limitation of RAP-PCR --- p.110 / Chapter 2.5.3 --- Two rounds of dot blot hybridization screening --- p.111 / Chapter CHAPTER THREE... --- CHARACTERIZATION OF THE ISOLATED GENE FRAGMENTS / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.1.1 --- Automated DNA sequencing and analysis --- p.113 / Chapter 3.1.2 --- GenBank and the BLAST homology search --- p.115 / Chapter 3.2 --- Materials --- p.118 / Chapter 3.2.1 --- Selected recombinant plasmids --- p.118 / Chapter 3.2.2 --- Chemicals --- p.118 / Chapter 3.2.3 --- Reagents --- p.118 / Chapter 3.2.4 --- Kits --- p.119 / Chapter 3.2.5 --- Solutions --- p.119 / Chapter 3.2.6 --- Equipment --- p.119 / Chapter 3.3 --- Methods --- p.120 / Chapter 3.3.1 --- Preparation of selected recombinant plasmid DNA --- p.120 / Chapter 3.3.2 --- Restriction digestion of recombinant plasmid DNA --- p.120 / Chapter 3.3.3 --- Automated DNA sequencing --- p.120 / Chapter 3.3.3.1 --- Primer annealing to template --- p.120 / Chapter 3.3.3.2 --- Sequencing reactions --- p.121 / Chapter 3.3.3.3 --- Polyacrylamide gel electrophoresis --- p.121 / Chapter 3.3.3.4 --- Data analysis by ALF manager and DNAsis --- p.122 / Chapter 3.3.4 --- Sequence homology search with databases --- p.122 / Chapter 3.4 --- Results --- p.123 / Chapter 3.4.1 --- Spectrophotometric analysis of selected recombinant plasmid DNAs subcloned with differentially amplified fragments --- p.123 / Chapter 3.4.2 --- Restriction digestion of selected recombinant plasmid DNA --- p.124 / Chapter 3.4.3 --- Sequences of the subcloned differentially amplified fragments --- p.126 / Chapter 3.4.4 --- Sequence analysis of the subcloned differentially amplified fragments --- p.144 / Chapter 3.5 --- Discussion --- p.157 / Chapter 3.5.1 --- Sequence analysis of the isolated gene fragment --- p.157 / Chapter CHAPTER FOUR … --- "EXPRESSION PROFILE OF ISOLATED GENES FRAGMENTS IN MYELOID LEUKEMIA CELL, MOUSE EMBRYO, AND TISSUES" / Chapter 4.1 --- Introduction --- p.162 / Chapter 4.1.1 --- Quantitation of mRNA by Reverse transcription-polymerase chain reaction --- p.162 / Chapter 4.1.2 --- Internal primer design by OLIGO´ёØ ver 34 --- p.167 / Chapter 4.2 --- Materials --- p.168 / Chapter 4.2.1 --- Mice --- p.168 / Chapter 4.2.2 --- Cell lysate --- p.168 / Chapter 4.2.3 --- Total RNAs --- p.168 / Chapter 4.3 --- Methods --- p.169 / Chapter 4.3.1 --- Internal primer design by OLIGO´ёØ ver 34 --- p.169 / Chapter 4.3.2 --- "Isolation of total RNA from biochanin A induced JCS cells, mouse embryos and tissue" --- p.169 / Chapter 4.3.2.1 --- Preparation of cell lysate from mouse embryo and postnatal mouse brain --- p.169 / Chapter 4.3.2.2 --- Isolation of RNA by guanidium thiocyanate cesium chloride method --- p.170 / Chapter 4.3.3 --- Preparation of saggital section of mouse embryo --- p.170 / Chapter 4.3.4 --- Confirmation of differential expression of isolated genes fragments during biochanin A and midazolam induced WEHI 3B (JCS) differentiation and the expression profile in mouse tissues and during mouse embryo development by reverse transcription-polymerase chain reaction --- p.171 / Chapter 4.4 --- Results --- p.173 / Chapter 4.4.1 --- Internal primer design of the sequenced fragments --- p.173 / Chapter 4.4.2 --- Spectrophotometric analysis of total RNA --- p.175 / Chapter 4.4.3 --- Saggital section of mouse embryo --- p.176 / Chapter 4.4.4 --- Normalization of RNA samples --- p.180 / Chapter 4.4.5 --- Analysis of mRNA expression of differentially amplified fragmentsin biochanin A or midazolam induced JCS cells and mouse embryos by RT- PCR --- p.182 / Chapter 4.4.5.1 --- "Genes downregulated at 1 hour, 5 hours and 48 hours after biochanin A induction of JCS cells" --- p.183 / Chapter 4.4.5.2 --- Genes up-regulated at 48 hours after biochanin A induction --- p.183 / Chapter 4.4.5.3 --- Genes constitutively expressed during the course of biochanin A treatment --- p.184 / Chapter 4.4.5.4 --- Genes showing undetectable level of expression in biochanin A induced JCS cells --- p.184 / Chapter 4.4.6 --- Tissue expression of the biochanin A induced-differentially expressed fragments by RT-PCR --- p.188 / Chapter 4.5 --- Discussion --- p.191 / Chapter 4.5.1 --- Expression profiles of isolated differentially amplified fragments --- p.191 / Chapter 4.5.2 --- Comparison of the expression profiles of the isolated gene fragments analyzed by dot blot hybridization screening and RT-PCR --- p.197 / Chapter CHAPTER FIVE ... --- GENERAL DISCUSSION --- p.200 / REFERENCES --- p.207 / APPENDIX --- p.234

HOXA7 belongs to a family of homeobox transcription factors that are master regulators of cell differentiation, morphogenesis during embryonic development and cell proliferation. Dysregulation and non-nuclear localization of these proteins play a role in a large number of solid tumours, with reports of significant upregulation of HOXA7 in oral dysplasia. It is unclear whether HOXA7 induction in solid tumours is causative or if it is a result of oncogenic changes. In this thesis we studied its effect on cell differentiation, growth, stemness, cell migration, EMT and cell senescence. The main hypothesis was that HOXA7 regulated keratinocyte differentiation through the regulation of activator protein 1 (AP-1), a keratinocyte specific activator of differentiation. We also hypothesised that HOXA7 increased the proliferation rate in keratinocytes. In an AP-1 reporter assay in HEK293 cells, HOXA7 was shown to decrease AP-1 activity significantly. The inactivation of AP-1 was not due to inactivation of PKC, as HOXA7 did not interfere with the activation of the kinases in HEK293. More specifically, we reported a very significant repression of c-Jun and JunD promoter activity in the presence of ectopic HOXA7 in HEK293 cells. We further showed that this mechanism might also be applicable in keratinocytes, as HOXA7 inhibited the transcription of AP-1 subunits of both the Jun and Fos family in skin keratinocytes. Furthermore, we showed transcriptional repression of four differentiation markers and a downregulation of K1 and FLG protein in transduced NEB-1 monolayers as well as K1 suppression in HaCaT cells. The organotypic cultures revealed a downregulation of K1, K10, and filaggrin in stratified HaCaT cells by HOXA7. There was however no downregulation in oral keratinocytes. These observations taken together suggested that HOXA7 repressed the synthesis of AP-1 units in skin keratinocytes, which would have resulted in reduced quantities of AP-1 and therefore lower activity. Contrary to previous reports, we observed no positive involvement of HOXA7 in keratinocyte proliferation, EMT or migration. There was however an indication of cell-type specific MET and induced cell senescence. Based on our results we propose a cell-type specific role of HOXA7 as an antagonist of AP-1 transcription in skin keratinocytes, and a possible direct binding of HOXA7 to c-Jun and JunD promoters.

L’apoptose, ou mort cellulaire programmée, joue un rôle majeur au cours du développement embryonnaire et dans le maintien de l’homéostasie tissulaire chez l’adulte. La voie mitochondriale de l’apoptose est principalement activée par la protéine oncosuppressive p53. Le FGF1 est un facteur de croissance atypique, majoritairement intracellulaire et nucléaire qui induit la prolifération, la différenciation et la survie cellulaires. Dans les cellules PC12, le FGF1 présente des activités neurotrophique et anti-apoptotique vis-à-vis de l’apoptose dépendante de p53. De plus, il interagit avec la protéine p53. La localisation nucléaire du FGF1 semble nécessaire à ses activités intracellulaires ainsi qu’à son interaction avec p53.Au cours de ma thèse, j’ai étudié les activités neurotrophique et anti-apoptotique de différentes formes mutantes du FGF1. J’ai entrepris l’étude de l’activité intracellulaire de la forme FGF1K132E, forme mutante dont les activités extracellulaires sont inhibées. La mutation du résidu lysine 132 pourrait modifier la phosphorylation du résidu sérine 130 du FGF1, j’ai donc également étudié deux autres formes mutantes : le FGF1S130A, dont la phosphorylation est inhibée et le FGF1S130D dont la phosphorylation est mimée. Les résidus mutés (K132 et S130) sont situés dans le domaine C-terminal du FGF1.Cette étude nous a permis de montrer que la phosphorylation du FGF1 inhibe son activité anti-apoptotique mais ne modifie pas son activité neurotrophique, et que le domaine C-terminal du FGF1 est fortement impliqué dans la régulation de ses activités intracellulaires. Toutes ces formes mutantes sont capables d’être transloquées dans le noyau ce qui suggère que la localisation nucléaire du FGF1 soit nécessaire mais insuffisante pour ses activités intracellulaires. Par ailleurs, p53 peut interagir avec le FGF1WT et certaines formes mutantes, toutefois cette interaction n’est pas strictement corrélée à l’activité anti-apoptotique du FGF1, ce qui suggère l’existence d’autres régulations nucléaires qui restent à caractériser.Mes travaux ont permis pour la première fois de mettre en évidence le rôle de la phosphorylation du FGF1 et de son domaine C-terminal dans la régulation de ses activités intracellulaires. La poursuite de cette étude permettra de mieux caractériser le rôle nucléaire de ce facteur de croissance et de caractériser ses éventuelles interactions avec des protéines nucléaires comme p53 / Apoptosis, a form of programmed cell death, is required for embryonic development and tissue homeostasis. The mitochondrial pathway of apoptosis is mainly induced by p53, an oncosuppressor that acts as a transcription factor. FGF1 is one of the two prototypic members of the FGF family. Contrarily to most FGFs, FGF1 lacks a secretion peptide signal and acts mainly in an intracellular and nuclear manner. Intracellular FGF1 induces cell proliferation, differentiation and survival. In PC12 cells, FGF1 inhibits p53-induced apoptosis and interacts with p53. FGF1 nuclear localization seems to be required for its intracellular activities and its interaction with p53.To better characterize the FGF1 intracellular pathway, we studied neurotrophic and anti-apoptotic activities of several mutant forms of FGF1: the FGF1K132E that could affect FGF1 phosphorylation, and two phosphorylation-site mutant forms, i.e. the FGF1S130A (preventing phosphorylation) and the FGF1S130D (mimicking phosphorylation). All these mutations are localized in the C-terminal domain of FGF1. This study showed that phosphorylation inhibits FGF1 anti-apoptotic activity but not its neurotrophic activity in PC12 cells and that the FGF1 C-terminal domain is strongly involved in the regulation of its intracellular activities. Despite their different activities, all mutant forms are localized both in the cytosol and the nucleus. Therefore, nuclear localization is required but insufficient for FGF1 to display its intracellular activities.Besides, p53 can interact with wild-type and some of the mutant forms of FGF1. This interaction does not strictly correlate with FGF1 anti-apoptotic activity. Thus, the nuclear mechanisms regulating FGF1 intracellular activities remain to be characterized.Our study highlights for the first time the role of the phosphorylation and the C-terminal domain of FGF1 on the regulation of its intracellular activities. This work must continue on to further characterize FGF1 nuclear activities and its interactions with nuclear proteins such as p53

Type 2 Diabetes (T2D) affects over 415 million people globally and is characterised by cellular stresses including: poor glucose homeostasis, dyslipidaemia, inflammation, hypoxia and ER stress. Studies in mice have shown that exposure to these stresses influences beta cell differentiation status as well as cell survival and may explain the extent of beta cell mass loss that is seen in the disease. To date, studies of altered beta cell differentiation have largely been confined to murine models. I used the EndoC-bH1 human beta cell line, along with human pancreatic tissue sections, to better characterise this mechanism in human disease. To elucidate these mechanisms, I firstly established a humanised version of cell culture techniques for the EndoC βH1 cell model and assessed the influence on cell function. Secondly, I evaluated the effects of the diabetic microenvironment on beta cell differentiation and gene expression patterns. Finally, I investigated whether a diabetomimetic microenvironment induced differences in microRNA regulation in the cells. I found that the humanised EndoC-βH1 culture techniques improved glucose sensitive insulin release in the cell model. EndoC-βH1 cells exposed to a Diabetic microenvironment showed some degree of transdifferentiation and this may be due to dysregulation of splicing factor expression. These effects may be compounded by altered microRNA regulation in response to these cell stresses. These data suggest that altered gene regulation caused by a diabetic microenvironment may alter gene regulation to produce a reversible delta-like phenotype in human beta cells.