Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 14, 2009). Includes bibliographical references.

Thesis (Ph. D. in Biological Sciences)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: histone deacetylase. Includes bibliographical references (p.35-36).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 193-218). Also available in electronic version.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Kathrin Schrick / Class IV homeodomain leucine-zipper transcription factors (HD-Zip TFs) are master regulators of cell-type differentiation in the plant epidermis. These transcription factors contain a putative START (STeroidogenic Acute Regulatory (StAR)-related lipid Transfer) lipid/sterolbinding domain that is hypothesized to link metabolism to gene expression in plant development. This study is focused on two class IV family members that serve as models in many of the experiments: GLABRA2 (GL2) is a key regulator of differentiation in hair cells called trichomes as well as other epidermal cell types in various plant tissues. The second member addressed in this study is PROTODERMAL FACTOR2 (PDF2), which plays a crucial role in epidermal cell specification in shoots. A leading hypothesis is that the START domain, by binding a ligand, controls transcription factor function, analogously to nuclear receptors from mammals. Domain swap experiments indicated that the START domain from both plants and mammals is a conserved ligand-binding motif that is required for transcription factor activity. To further address its function in ligand binding, mutational analysis of the START domain of GL2 was performed. Several of the mutations remove charged residues in the predicted ligand-binding pocket and resulted in loss-of-function phenotypes, suggesting that ligand binding is critical for HD-Zip TF activity. Chromatin immunoprecipitation–based sequencing (ChIP-seq) revealed that the START domain is dispensable for transcription factor binding to DNA. Using a high throughput thermal shift assay to screen a library of pure natural compounds, specific secondary metabolites were identified as putative START domain ligands for PDF2. Experiments in both yeast and N. benthamiana demonstrated that the START domain is required for homodimerization of GL2 through its Zip domain. It was also found that the START domains physically interact with RHAMNOSE SYNTHASE I (RHM1). Further, this work provided evidence for a previously elusive redundancy between GL2 and another class IV HD-Zip TF, and unveils a positive feedback loop in the maintenance of the GL2 activity during trichome differentiation. Taken together, these findings support the premise that START domains are central players in metabolic regulatory networks that can modulate transcription factor activity by binding ligands and mediating protein-protein interactions.

No description available.

The homeodomain transcription factor family of Cdx genes, Cdx1, Cdx2 and Cdx4, are known to play essential roles in many developmental processes including neural tube closure, axial elongation, hematopoiesis and gastrointestinal patterning. In the adult, Cdx1 and Cdx2 are both expressed strictly in the adult intestinal epithelium, but their functions and mechanisms of action at this stage are poorly understood. Cdx transcription factors have also been reported to be lost in intestinal cancers. To circumvent early lethality, a conditional loss of function strategy was used to inactivate Cdx2 in the adult intestinal epithelium. These conditional mutants were crossed to Cdx1-/- mice to examine potential functional compensation between these family members as well as into APC(min/+) mice to study their role in tumorigenesis. Using these models, I have found that Cdx2 regulates adult intestinal homeostasis and differentiation in the small intestinal epithelium, while both Cdx1 and Cdx2 contribute to colon homeostasis. Furthermore, Cdx transcription factors are tumor suppressors in the development of Wnt-induced colorectal cancer, and impact several pathways including TGF-β and Eph-ephrin signaling. Finally, Cdx2 regulates Eph-ephrin signaling through direct activation of the Notch pathway. Altogether, this study underscores critical roles and mechanisms of action for Cdx members in the adult intestine and in intestinal tumorigenesis.

Transcription factors (TFs) are proteins that respond to a specific chemical signal and bind to DNA. In some bacteria, TFs control transition metal ion homeostasis by specifically binding with a particular metal ion or ions and then interacting with DNA. Although most first row metal ions are required as micronutrients for life, many of them can cause cellular damage or death if their concentrations are too high; this makes these TFs and their biological interactions excellent targets for drug development. The bacteria, Streptococcus pneumoniae is a pathogenic microorganism responsible for a range of diseases that target the young and the old, including pneumonia, meningitis, and bacteremia. Herein, we describe our efforts to study the Streptococcus pneumoniae TF responsible for copper(II) ion homeostasis. This thesis describes the classical biochemical techniques used to over-express and purify CopR. It also describes a series of preliminary characterization data associated with this novel copper(II)-dependent TF.

Tsui, Wing Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 125-137). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / Chinese abstract --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.X / List of figures --- p.xiv / List of tables --- p.XV / Table of contents --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Literature review on LIM-homeobox genes in mouse development --- p.1 / Chapter 1.1.1 --- LIM-homeobox genes --- p.1 / Chapter 1.1.2 --- Mouse Lhx1 gene and development --- p.5 / Chapter 1.1.3 --- Mouse Lhx5 gene and development --- p.21 / Chapter 1.2 --- Mouse cerebellar Purkinje neurons --- p.26 / Chapter 1.2.1 --- Cerebellar cortex --- p.26 / Chapter 1.2.2 --- Neuronal circuitry and cerebellar functions --- p.29 / Chapter 1.2.3 --- Development of cerebellar Purkinje neurons --- p.29 / Chapter 1.2.3.1 --- Neurogenesis --- p.30 / Chapter 1.2.3.2 --- Migration and positioning --- p.30 / Chapter 1.2.3.3 --- Specification and differentiation --- p.31 / Chapter 1.2.3.4 --- Maturation --- p.31 / Chapter 1.3 --- Green Fluorescent Protein (GFP) and tau protein --- p.32 / Chapter 1.3.1 --- Introduction to tau proteins --- p.32 / Chapter 1.3.2 --- Tau-GFP fusion protein and its application in tracing neuronal projections --- p.33 / Chapter 1.4 --- Project background and aim --- p.34 / Chapter Chapter 2 --- Generation of Lhx1-tau-GFP knock-in mice --- p.38 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.2 --- Materials for molecular biological work --- p.39 / Chapter 2.2.1 --- Chemicals and kits --- p.39 / Chapter 2.2.2 --- Enzymes --- p.40 / Chapter 2.2.3 --- Plasmid vectors --- p.40 / Chapter 2.2.4 --- Oligonucleotide linkers --- p.41 / Chapter 2.2.5 --- Bacterial strains --- p.41 / Chapter 2.2.6 --- Solutions and media --- p.41 / Chapter 2.2.7 --- Radioactive isotopes and materials for autoradiography --- p.43 / Chapter 2.2.8 --- DNA probes for Southern blot hybridization --- p.43 / Chapter 2.3 --- Materials for cell culture --- p.44 / Chapter 2.3.1 --- "Chemicals, sera and others" --- p.44 / Chapter 2.3.2 --- Culture solutions and media --- p.44 / Chapter 2.3.3 --- Culture cells --- p.45 / Chapter 2.4 --- PCR primers --- p.46 / Chapter 2.5 --- Animals --- p.46 / Chapter 2.6 --- Methods for molecular biological work --- p.46 / Chapter 2.6.1 --- Preparation of plasmid DNA --- p.46 / Chapter 2.6.1.1 --- Miniprep using simple crude method --- p.47 / Chapter 2.6.1.2 --- Miniprep using purification kits --- p.48 / Chapter 2.6.1.3 --- Midiprep using purification kit --- p.50 / Chapter 2.6.2 --- Purification of specific DNA fragments --- p.51 / Chapter 2.6.2.1 --- QIAquick gel extraction kit --- p.51 / Chapter 2.6.2.2 --- QIAquick PCR purification kit --- p.52 / Chapter 2.6.3 --- Subcloning of DNA fragments --- p.53 / Chapter 2.6.3.1 --- Traditional approach based on restriction endonuclease and DNA ligase --- p.53 / Chapter 2.6.3.2 --- Preparation of subcloning inserts and vectors --- p.54 / Chapter 2.6.3.3 --- Two-way ligation of inserts and vectors --- p.55 / Chapter 2.6.4 --- Transformation of competent cells with recombinant DNA --- p.56 / Chapter 2.6.4.1 --- CaCl2 method --- p.56 / Chapter 2.6.4.2 --- Electroporation --- p.57 / Chapter 2.6.5 --- Southern hybridization --- p.59 / Chapter 2.6.5.1 --- Restriction endonuclease digestion and agarose gel electrophoresis --- p.59 / Chapter 2.6.5.2 --- Capillary transfer and fixation of DNA --- p.60 / Chapter 2.6.5.3 --- Radioactive labeling of DNA probe --- p.60 / Chapter 2.6.5.4 --- Purification of radioactive labeled probe for hybridization --- p.61 / Chapter 2.6.5.5 --- Hybridization --- p.61 / Chapter 2.6.5.6 --- Post-hybridization wash and autoradiography for signal detection --- p.62 / Chapter 2.7 --- Methods for generation and analysis of Lhx1-tau-GFP knock-in Mice --- p.63 / Chapter 2.7.1 --- Construction of targeting vector (pLhx1-tauGFP) for gene targeting of Lhx1 locus --- p.63 / Chapter 2.7.2 --- Generation of targeted embryonic stem (ES) cell clones --- p.66 / Chapter 2.7.2.1 --- Preparation of feeder cells --- p.66 / Chapter 2.7.2.2 --- Culture of ES cells on feeder layers and passage --- p.69 / Chapter 2.7.2.3 --- Harvest of cultured ES cells --- p.70 / Chapter 2.7.2.4 --- Preparation of targeting vector for transfection of ES cells --- p.71 / Chapter 2.7.2.5 --- Electroporation for transfection of ES cells --- p.71 / Chapter 2.7.2.6 --- Drug selection for targeted ES cell clones using PNS strategy --- p.72 / Chapter 2.7.2.7 --- Picking and expansion of targeted ES cell clones --- p.72 / Chapter 2.7.2.8 --- Replica plating and freezing of targeted ES cell clones --- p.74 / Chapter 2.7.2.9 --- Genomic DNA extraction from targeted ES cell clones --- p.75 / Chapter 2.7.2.10 --- Screening of homologous recombinants by Southern hybridization analysis --- p.76 / Chapter 2.7.2.11 --- Thawing and expansion of correct targeted ES cell clones --- p.76 / Chapter 2.7.2.12 --- Chromosome counting of ES cells --- p.78 / Chapter 2.7.3 --- Generation of germline chimeric mice --- p.80 / Chapter 2.7.3.1 --- Standard procedure --- p.80 / Chapter 2.7.4 --- Breeding and genotyping of mice --- p.81 / Chapter 2.7.5 --- Imaging of tau-GFP-labelled Purkinje neurons --- p.84 / Chapter 2.7.5.1 --- Animal dissection and tissue preparation --- p.84 / Chapter 2.7.5.2 --- Confocal laser scanning microscopy (CLSM) --- p.84 / Chapter 2.8 --- Results --- p.84 / Chapter 2.8.1 --- Generation of Lhx1 targeting vector (pLhx1-tauGFP) --- p.84 / Chapter 2.8.2 --- Targeted replacement of the mouse Lhx1 coding sequences by tau-GFP genetic reporter --- p.87 / Chapter 2.8.3 --- Germline transmission of Lhx1-tau-GFP allele and generation of Lhx1-tau-GFP knock-in mouse --- p.93 / Chapter 2.8.4 --- Imaging of Lhx1-tau-GFP expressing Purkinje neurons --- p.96 / Chapter 2.9 --- Discussion --- p.98 / Chapter 2.9.1 --- Tau-GFP labeling of Lhx1-expressing Purkinje neurons: implications for real-time live cell imaging --- p.98 / Chapter 2.9.2 --- Use of Lhx1-tau-GFP knock-in mice for study of Lhx1 and Lhx5 functions in Purkinje neurons survival and/or maintenance --- p.99 / Chapter Chapter 3 --- Generation of Lhx5-tau-GFP knock-in allele: alternative approach for real-time tracing of Purkinje neurons --- p.102 / Chapter 3.1 --- Introduction: Recombineering-based approach for DNA subcloning --- p.102 / Chapter 3.1.1 --- λ phage-encoded Red recombination system --- p.102 / Chapter 3.1.2 --- DNA subcloning from bacterial artificial chromosome (BAC) --- p.104 / Chapter 3.2 --- Materials for molecular biological work --- p.105 / Chapter 3.2.1 --- Chemicals and kits --- p.105 / Chapter 3.2.2 --- Enzymes --- p.105 / Chapter 3.2.3 --- Plasmid vectors and BAC DNA --- p.105 / Chapter 3.2.4 --- Bacterial strains --- p.105 / Chapter 3.2.5 --- Solutions and media --- p.106 / Chapter 3.2.6 --- PCR primers --- p.106 / Chapter 3.3 --- Methods for construction of targeting vector for mouse Lhx5 gene --- p.107 / Chapter 3.3.1 --- PCR amplification of homology sequences on BAC DNA --- p.107 / Chapter 3.3.2 --- Synthesis of retrieval arms for recombineering --- p.109 / Chapter 3.3.3 --- DNA sequencing analysis --- p.110 / Chapter 3.3.4 --- Construction of retrieval vector --- p.110 / Chapter 3.3.5 --- Preparation of electrocompetent cells for recombineering --- p.111 / Chapter 3.3.6 --- Recombineering-based retrieval of homology arms --- p.112 / Chapter 3.4 --- Results --- p.113 / Chapter 3.4.1 --- The targeting vector (pLhx5-tauGFP) for mouse Lhx5 gene --- p.113 / Chapter 3.5 --- Discussion --- p.118 / Chapter 3.5.1 --- Use of recombineering-based approach to generate targeting vector --- p.118 / Chapter 3.5.2 --- Further generation of Lhx5-tau-GFP knock-in mice --- p.119 / Chapter Chapter 4 --- Conclusion and future perspectives --- p.120 / Chapter 4.1 --- Conclusion --- p.120 / Chapter 4.2 --- Potential applications of Lhx1-tau-GFP knock-in mice for study of Lhx1 and other gene functions in cerebellum --- p.120 / Chapter 4.3 --- Potential applications of Lhx1-tau-GFP knock-in mice for study of Lhx1 -expressing cells development --- p.122 / References --- p.125

Chan, Pui Ting. / Thesis submitted in: May 2007. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 110-118). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Peroxisome proliferater-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR isoforms --- p.1 / Chapter 1.1.3 --- PPARα ligands --- p.2 / Chapter 1.2 --- Biological role of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.5 / Chapter 1.2.3 --- Oxidative stress and carcinogenesis --- p.6 / Chapter 1.3 --- Discovery of PPARα-regulated and starvation-induced gene (PPSIG) --- p.7 / Chapter 1.4 --- Objectives of the present study --- p.9 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Cloning of PPSIG cDNA into a pCMV-Tag epitope tagging mammalian expression vector --- p.10 / Chapter 2.1.1 --- Materials --- p.10 / Chapter 2.1.2 --- Methods --- p.10 / Chapter 2.2 --- Transient transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.16 / Chapter 2.2.1 --- Cell culture and transfection --- p.16 / Chapter 2.2.1.1 --- Materials --- p.16 / Chapter 2.2.1.2 --- Methods --- p.19 / Chapter 2.2.2 --- Western blot analysis --- p.20 / Chapter 2.2.2.1 --- Materials --- p.20 / Chapter 2.2.2.2 --- Methods --- p.20 / Chapter 2.3 --- Stable transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.22 / Chapter 2.3.1 --- Linearization of the pCMVT4B-PPSIG construct --- p.22 / Chapter 2.3.1.1 --- Materials --- p.22 / Chapter 2.3.1.2 --- Methods --- p.22 / Chapter 2.3.2 --- Cell culture and stable transfection --- p.23 / Chapter 2.3.2.1 --- Materials --- p.23 / Chapter 2.3.2.2 --- Methods --- p.23 / Chapter 2.3.3 --- Selection of the G418-resistant clones --- p.26 / Chapter 2.3.3.1 --- Materials --- p.26 / Chapter 2.3.3.2 --- Methods --- p.29 / Chapter 2.3.4 --- Picking and expanding the G418-resistant clones --- p.30 / Chapter 2.3.4.1 --- Materials --- p.30 / Chapter 2.3.4.2 --- Methods --- p.30 / Chapter 2.3.5 --- Screening and confirmation of the stable transfectants --- p.31 / Chapter 2.3.5.1 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.31 / Chapter 2.3.5.1.1 --- Materials --- p.31 / Chapter 2.3.5.1.2 --- Methods --- p.31 / Chapter 2.3.5.2 --- Northern blot analysis --- p.35 / Chapter 2.3.5.2.1 --- Materials --- p.35 / Chapter 2.3.5.2.2 --- Methods --- p.35 / Chapter 2.3.5.3 --- Western blot analysis --- p.37 / Chapter 2.3.5.3.1 --- Materials --- p.37 / Chapter 2.3.5.3.2 --- Methods --- p.37 / Chapter 2.3.5.4 --- Immunoprecipitation --- p.37 / Chapter 2.3.5.4.1 --- Materials --- p.37 / Chapter 2.3.5.4.2 --- Methods --- p.38 / Chapter 2.3.5.5 --- Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry analysis --- p.39 / Chapter 2.3.5.5.1 --- Materials --- p.39 / Chapter 2.3.5.5.2 --- Methods --- p.39 / Chapter 2.4 --- "Analysis of the all-trans-13,14-dihydroretinol saturase (RetSat) activity by high-performance liquid chromatography (HPLC) analysis" --- p.41 / Chapter 2.4.1 --- Materials --- p.41 / Chapter 2.4.2 --- Methods --- p.42 / Chapter 2.4.2.1 --- Preparation of all-trans-retinol --- p.42 / Chapter 2.4.2.2 --- Treatment of PPSIG-transfected cells with all-trans-retinol --- p.42 / Chapter 2.4.2.3 --- Retinoid analysis --- p.43 / Chapter 2.5 --- Analysis of fatty acid compositions by gas chromatography-mass spectrometry (GC-MS) --- p.43 / Chapter 2.5.1 --- Materials --- p.43 / Chapter 2.5.2 --- Methods --- p.44 / Chapter 2.5.2.1 --- Preparation of fatty acid-BSA complex --- p.44 / Chapter 2.5.2.2 --- Treatment of PPSIG-transfected cells with fatty acid-BSA complex --- p.44 / Chapter 2.5.2.3 --- Extraction of fatty acids --- p.45 / Chapter 2.5.2.4 --- Methylation of the fatty acids --- p.45 / Chapter 2.5.2.5 --- GC-MS analysis --- p.46 / Chapter 2.5.2.6 --- Statistical analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.48 / Chapter 3.1 --- The PPSIG cDNA was subcloned into a pCMV-Tag epitope tagging mammalian expression vector --- p.48 / Chapter 3.2 --- The pCMVT4B-PPSIG expression construct was transiently transfected into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3 --- Stable transfection of the pCMVT4B-PPSIG expression construct into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3.1 --- PPSIG-transfected CHO-K1 and AML-12 cells were obtained after G418 selection --- p.54 / Chapter 3.3.2 --- PPSIG-transfected CHO-K1 and AML-12 cells had high PPSIG mRNA expression --- p.58 / Chapter 3.3.3 --- PPSIG-FLAG fusion protein was over-expressed in the PPSIG- transfected CHO-K1 and AML-12 cells --- p.61 / Chapter 3.3.4 --- The stable transfectants were immunoprecipitated and identified as PPSIG protein by the mass spectrometry analysis --- p.64 / Chapter 3.4 --- PPSIG protein posseses saturase activity towards all-trans-retinol --- p.66 / Chapter 3.5 --- PPSIG protein is not a fatty acid transporter --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.101 / FUTURE STUDIES --- p.107 / REFERENCES --- p.110 / Appendix A: Prediction of the molecular weight of pCMVT4B- PPSIG protein --- p.119 / Appendix B: Theoretical tryptic peptides of PPSIG --- p.120 / Appendix C: Protein-peptide mass reports --- p.122 / Chapter C1. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone L2H4B18 --- p.122 / Chapter C2. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone AL2L7 --- p.123 / Appendix D: HPLC spectrum of the RetSat activity towards all- trans retinol --- p.124 / Chapter D1. --- RetSat activity towards all-trans retinol according to the Moise's group study ((Moise et al. 2004) --- p.124