A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. 2015. / SNAMA, the protein of interest in this thesis is found in the common model organism Drosophila melanogaster, also known as the fruit fly it is also found in all eukaryotic organisms but not in prokaryotes. SNAMA is a 1231 amino acid protein that belongs to the RbBP6 superfamily. Members of this family are characterized by a zinc finger motif, a DWNN domain (domain with no name) and a RING finger motif. The human RbBP6 contains the Rb-binding and p53-binding domains in addition. The mammalian RbBP6 hence interacts with p53 and Rb and it is important for the development and tumorigenesis as a negative regulator of p53. Bioinformatics studies show that transcription of the Snama gene is driven by a single TATA-less promoter which give rise to a single 3.9 kb transcript. However, experimental evidence confirming the promoter region has not being published. The main aim of this study was to examine the regulation of Snama by identifying the maximal promoter sequence that shows promoter activity in mammalian cell line. This was achieved by using specifically designed primers to amplify the putative Snama promoters, ligating promoters in reporter vector (pGL3 basic). The recombinant products used to transfect eukaryotic cells (Cos7, African green monkey cells) and determining the maximal promoter sequence that expresses luciferase activity. The promoter sequences were labelled with biotin attached to the primers and Electrophoretic mobility shift assay (EMSA) was conducted to confirm binding of proteins on the putative promoter fragments. The segment designated promoter 6 has maximal positive activity and many proteins in the cell extract bind to it shown by EMSA. Interestingly the longer fragment designated promoter 7 has less promoter activity. This may suggest that this fragment also contains some repressive elements.

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

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Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisors: Patricia A. Martin-DeLeon and Deni S. Galileo, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 161-195). Also available in electronic version.

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

Vaccinia virus infected monkey kidney cells had been previously shown to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA from mutant virus resistant to hydroxyurea were digested with restriction endonucleases and were shown to have substoichiometric amounts of the Hind III F fragment. Additional information from Southern blotting experiments localized the putative small subunit (R2) gene to the left end of the Hind III F fragment of the vaccinia virus genome. The entire open reading frame of the R2 gene and the flanking regions was sequenced and the translated sequence found to be 80% homologous to the mouse R2 polypeptide. A combination of in situ and in vitro experiments addressed the question of macromolecular interactions involving vaccinia ribonucleotide reductase (FIR). Replication of double stranded viral DNA occurs in very discrete loci in infected cells and these DNA factories can be isolated from gently lysed cell in sucrose gradients. RR was detected at low levels (less than 5% of the total R2) with the rapidly sedimenting DNA by using antibodies against FIR. In situ crosslinking experiments were attempted with no specific interaction determined at this time. Immunolocalization experiments have given evidence for localization of large subunit (R1) polypeptide to the viral inclusion bodies. The most conclusive results utilized anti-idiotypic antibodies against the antibodies to R2 protein. lmmunolocalization experiments have shown the putative R2 binding protein to be localized at the sites of viral DNA synthesis. lmmunoprecipitations show a single predominant viral polypeptide which also has proven to be a DNA binding (phospho)protein. Screening a lambda phale expression library of vaccinia with the anti-idiotypic antibody localized the binding site to the carboxy terminal 81 amino acids in open reading frame 1-3 of the vaccinia genome. The open reading frame was cloned into a pET11c expression vector and the partially purified recombinant protein was shown to have specificity for single-stranded DNA as well as stimulate vaccinia RR activity. / Graduation date: 1993

The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 10, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 243-262).