The extracellular matrix (ECM) plays an important role in mammary gland development and function. Osteopontin (OPN), a secreted glycophosphoprotein, with several functional and structural properties of ECM proteins, is expressed at elevated levels during normal and pathologic development of the mammary gland and is present in milk. However, whether it plays any developmental role in the mammary gland is unknown. To investigate this possibility, transgenic AS-OPN mice were generated using a transgene expressing OPN antisense RNA under the control of the MMTV-LTR promoter/enhancer. The mammary glands of AS-OPN mice express low levels of OPN, compared to control littermates, show excessive branching of the ductal epithelium at the virgin stage, impaired proliferation of the mammary epithelium, and abnormal alveolar structure formation during pregnancy, and mild to severe lactation deficiency. To further examine the role of OPN at the cellular level, an established murine mammary epithelial cell line (NMuMG), which is capable of differentiation in culture, was transfected with the same DNA construct used to generate AS-OPN mice. Several antisense OPN cDNA transfected clones, which secrete decreased amounts of OPN compared to mock-transfected cells were obtained. These cells were found to have lost their ability to form branched duct-like structures, as judged by branching morphogenesis assays, by growth in collagen gels and stimulation with hepatocyte growth factor. They also failed to spread on type I collagen, although their binding to type IV collagen was unaffected. The antisense transfectants also assumed a mesenchymal phenotype, characterized by fibroblast-like morphology, an apparent loss of cell-cell contacts and spontaneous cell scattering. Transmigration assays and wounding experiments indicated that these cells also have a higher migratory activity than control cells. Northern blot and immunofluorescence analyses showed that migrating cells downregulate OP

Mutations in the p53 gene are known to be associated with a wide range of human tumors. In some primary tumors and established cell lines, stable mutant p53 protein is expressed at high levels, whereas, in normal cells unstable wild-type p53 protein is expressed at very low levels. Sera from some patients with breast and colon tumors contain anti-p53 antibodies. It is unclear whether changes in p53 structure, or its increased level in tumors, causes p53 to become antigenic. In our study we tested sera from patients with various types of cancer for anti-p53 antibodies. Examination of the sera was made by Western blot, and the results were confirmed by rescreening sera with immunoprecipitation. Both techniques revealed the presence of anti-p53 antibodies in some sera from lung and ovary cancer patients, as well as in the sera from patients with breast or colon cancers. Clearly, patients with various cancer tumors are able to produce anti-p53 antibodies. It was unclear whether this humoral immune response is against mutant or wild type p53. To provide a better definition of this immune response, we have examined the anti-p53 response from cancer patients against mutant and wild type p53 in the native and denaturated state. Western blot and Immunoprecipitation analysis showed that the anti-p53 sera recognise both wild type and mutant p53 conformational and denaturation resistant epitopes. Taken together, these data demonstrate that the mutant p53 is not more antigenic than the wild type p53. This provides strong evidence that the antibody response is not directed solely against the altered conformation in mutant p53.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

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Chan Kam Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 175-182). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axis in Drosophila --- p.2 / Chapter 1.3 --- The maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.11 / Chapter 1.5 --- The formation of the biocid protein gradient from anterior pole to posterior pole of the embryo --- p.13 / Chapter 1.6 --- The bed protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.15 / Chapter 1.7 --- The formation of the bed protein gradient in embryo --- p.17 / Chapter 1.8 --- Components required for bcd mRNA localization at anterior pole of oocyte --- p.21 / Chapter 1.8.1 --- Cis-acting elements --- p.21 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.9 --- The properties of exuperantia protein --- p.25 / Chapter 1.9.1 --- The function of exu protein --- p.25 / Chapter 1.9.2 --- Exuperantia is a phosphoprotein --- p.26 / Chapter 1.9.3 --- Phosphorylation pattern of exuperantia protein is stage-specific --- p.28 / Chapter 1.9.4 --- Reversible phosphorylation is one of the major mechanisms to control protein activity in all eukaryotic cells --- p.29 / Chapter 1.9.5 --- The relationship between the exu protein phosphorylation and the bcd mRNA localization --- p.30 / Chapter 1.10 --- Aim of project --- p.31 / Chapter CHAPTER 2 --- Preparation of the exuperantia genomic DNA and complement DNA (cDNA) mutant Constructs / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- DNA preparation methods --- p.35 / Chapter 2.2.1.1 --- Preparation of double-stranded DNA by polyethylene glycol6000 --- p.35 / Chapter 2.2.1.2 --- Preparation of M13mp8 single-stranded DNA --- p.37 / Chapter 2.2.1.3 --- "Preparation of double-stranded DNA by Biol prep (Modified from Maniatis et al.,1989)" --- p.38 / Chapter 2.2.2 --- "Preparation of DH5α，JM109, TG1 competent cells" --- p.39 / Chapter 2.2.3 --- Bacteria transformation --- p.40 / Chapter 2.2.4 --- Restriction enzyme digestion --- p.40 / Chapter 2.2.5 --- Phenol/chloroform extraction --- p.41 / Chapter 2.2.6 --- Purification of DNA fragment by electro-elution --- p.42 / Chapter 2.2.7 --- DNA ligation --- p.43 / Chapter 2.2.8 --- DNA dephosphorylation --- p.43 / Chapter 2.2.9 --- In vitro site-directed mutagenesis --- p.44 / Chapter 2.2.9.1 --- The Sculptor´ёØ in vitro mutagenesis --- p.44 / Chapter 2.2.9.2 --- The GeneEditor´ёØ in vitro site-directed mutagenesis --- p.47 / Chapter 2.2.10 --- The double-stranded or single-stranded DNA sequencing by T7 DNA polymerase sequencing system --- p.50 / Chapter 2.2.11 --- Denatured polyacrylamide gel electorphoresis --- p.51 / Chapter 2.2.11 --- Nucleotide sequence of the sequencing primers and the mutageneic oligonucleotides --- p.54 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Design exuperantia mutant constructs --- p.55 / Chapter 2.3.1.1 --- Comparison of exu protein amino acids sequence with different Drosophila species --- p.56 / Chapter 2.3.2 --- The exu genomic mutant constructs --- p.63 / Chapter 2.3.3 --- The exu cDNA mutant constructs --- p.63 / Chapter 2.4 --- Discussion --- p.76 / Chapter CHAPTER 3 --- Epitope tagging of exuperantia protein with c-myc eptiope / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and methods --- p.84 / Chapter 3.2.1 --- Preparation of the c-myc eptiope DNA fragment --- p.84 / Chapter 3.2.2 --- End-filling of 5'overhang DNA fragment by Klenow fragment --- p.86 / Chapter 3.2.3 --- In vitro translation of protein by TNT® Quick coupled transcription and translation system --- p.86 / Chapter 3.2.4 --- Immunoprecipitation of recombinant exu protein --- p.87 / Chapter 3.2.5 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 3.2.5.1 --- SDS-PAGE preparation --- p.88 / Chapter 3.2.5.2 --- SDS-PAGE electrophoresis --- p.90 / Chapter 3.2.6 --- Western blot analysis --- p.90 / Chapter 3.2.6.1 --- Transfer the protein to a nitro-cellulose membrane by semi-dried blotting --- p.90 / Chapter 3.2.6.2 --- Western blot blocking and antibody recognition --- p.91 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Construction of the plasmid containing exu cDNA tagging with a c-myc epitope --- p.92 / Chapter 3.3.2 --- In vitro translation of c-myc epitope tagged exu protein --- p.102 / Chapter 3.3.3 --- Immunoprecipitation of c-myc labeled exu protein by a polyclonal rabbit anti-exu antibody and monoclonal mouse anti-myc antibody --- p.104 / Chapter 3.4 --- Discussion --- p.109 / Chapter CHAPTER 4 --- In vitro phosphorylation of exuperantia Protein / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and methods --- p.113 / Chapter 4.2.1 --- Exogenous kinase phsophorylation reactions --- p.113 / Chapter 4.2.2 --- Separation of the phosphorylated exu protein variants by SDS- PAGE --- p.114 / Chapter 4.3 --- Results --- p.115 / Chapter 4.3.1 --- Western blot analysis of in vitro translated exu protein variants --- p.115 / Chapter 4.3.2 --- Phosphorylation of in vitro translated exu protein variants by exogenous cAMP-dependent protein kinase --- p.118 / Chapter 4.3.3 --- Phosphorylation of in vitro translated exu protein variants by exogenous cGMP-dependent protein kinase --- p.123 / Chapter 4.3.4 --- Phosphorylation of in vitro translated exu protein variants by exogenous protein kinase C --- p.128 / Chapter 4.4 --- Discussion --- p.133 / Chapter CHAPTER 5 --- Introduction of the exuperantia genomic constrcuts into the germline of Drosophila by P element-mediated transformation / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Materials and methods --- p.138 / Chapter 5.2.1 --- Construction of a genomic construct for production of transgenic flies --- p.138 / Chapter 5.2.2 --- Preparation of double-stranded DNA by ultra-centrifugation --- p.142 / Chapter 5.2.3 --- P-element mediated transformation --- p.143 / Chapter 5.2.3.1 --- Eggs collection --- p.143 / Chapter 5.2.3.2 --- Dechorionating the eggs --- p.143 / Chapter 5.2.3.3 --- Orientating the eggs --- p.144 / Chapter 5.2.3.4 --- Microinjection --- p.145 / Chapter 5.2.4 --- Collecting virgin female Drosophila --- p.146 / Chapter 5.2.5 --- Setup a crossing experiment --- p.146 / Chapter 5.2.6 --- Preparation of total ovaries and testes extracts exu protein from Female and male Drosophila --- p.147 / Chapter 5.2.7 --- Immunohistochemical distribution of exuperantia protein --- p.147 / Chapter 5.3 --- Results --- p.150 / Chapter 5.3.1 --- Insertion of the mutated exu fragments into the Drosophila Transformation vector (pCaSpeR) --- p.150 / Chapter 5.3.2 --- Introduction of the mutated exu gene into the genome of Drosophila by P-element mediated transformation --- p.153 / Chapter 5.3.3 --- Western blot analysis of the exu protein in the exu (ES2.1) transgenic fly --- p.160 / Chapter 5.3.4 --- Immunohistochemical distribution of exu protein in exuES21 mutants --- p.162 / Chapter 5.3.5 --- Rescue test of exuES2.1 trangenic flies --- p.165 / Chapter 5.4 --- Discussion --- p.168 / Chapter CHAPTER 6 --- General Discussion --- p.171 / References --- p.173 / Chapter Appendix I: --- List of reagents --- p.183 / Chapter Appendix II: --- Publication --- p.187

Diatoms are unicellular photosynthetic algae that display intricately patterned cell walls made of amorphous silicon dioxide (silica). Long-chain polyamines and highly phosphorylated proteins, silaffins and silacidins, are believed to play an important role in biosilica formation. The phosphate moieties on silaffins and silacidins play a significant role in biomineral formation, yet no kinase has been identified that phosphorylates these biomineral forming proteins. This dissertation describes the characterization of a novel kinase from the diatom Thalassiosira pseudonana, tpSTK1, which is upregulated during silica formation. A recombinantly expressed histidine-tagged version of tpSTK1 was capable of phosphorylating recombinant silaffins but not recombinant silacidin in vitro. Through establishing methods for subcellular fraction of T. pseudonana membranes in combination with antibody inhibition assay, it was discovered that native tpSTK1 phosphorylates silaffins but not silacidins in vitro (i.e. it exhibits the same substrate specificity as recombinant tpSTK1). As tpSTK1 is an abundant protein in the ER lumen (~ 0.5 % of total ER protein) it seems highly likely to function as a silaffin kinase in vivo. TpSTK1 lacks clear sequence homologs in non-diatom organisms and is the first molecularly characterized kinase that appears to be involved in biomineralization. The predicted kinase domain (KD) of tpSTK2, the only T. pseudonana homolog of tpSTK1, was recombinantly expressed and tested for phosphorylation activity. Recombinant tpSTK2-KD and native tpSTK2 exhibited detectable activity with myelin basic protein, but did not phosphorylate silaffins or silacidins in vitro. Western blot analysis demonstrated that native tpSTK2 was not present in the ER, but associated with the cytosol and Golgi membrane containing subcellular fractions.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 91-109).

DNA replication is cruciaI for the transmission of genetic information. Understanding the enzymology involved in this complex process will allow further insight into its mechanism. Experimental evidence indicates a role for DNA ligase I in DNA replication. Techniques of molecular and cellular biology and immunology were utilized in this study to further investigate DNA ligase I and clarify its involvement and interaction with other proteins in DNA replication. Immunofluorescence studies were performed to examine the intracellular distribution of DNA ligase I. Confocal analysis of indirect immunofluorescence detection of DNA ligase I using affinity purified anti-human DNA ligase I antibodies showed nuclear localization of DNA ligase I in distinct foci resembling those structures seen in detection of centres of DNA replication and other DNA replication proteins. Immunoprecipitation experiments were performed on extracts of MDBK cells to examine possible interactions of DNA ligase I with the DNA replication cofactor, PCNA; and no interactions were detected.