Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Abstracted in Dissertation abstracts, v. 16 (1956) no. 11, p. 2010-2011. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 32-35).

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 90-110). Also available on the World Wide Web.

The Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.

Cells exposed to DNA damaging agents activate a network of mechanisms called DNA damage response, including telomere length regulation. Telomeres are specialized structures that protect chromosome ends from degrading and being fused together. Mouse-knockout experiments revealed that cell lines deficient of DNA-PKcs or Ku70/80 resulted in high amount of telomere end-to-end fusion. Numerous other studies have shown a functional interplay between DNA damage response and telomere maintenance. The aim of this project is to examine this interplay further by investigating mechanisms of DNA damage response, using cell lines from X-linked homozygous recessive form of Dyskeratosis Congenita (DC) patients, which have dysfunctional telomere maintenance. DC is a multi-system disorder characterised by abnormalities of the bone marrow, immune deficiency and a predisposition to cancer. In this work we have shown that cells with defective DKC1 (the gene implicated in the X- linked homozygous recessive form of DC) exhibit a defective DNA damage response by examining two types of cells: fibroblast and lymphoblastoid cell lines. By using various biomarkers (H2AX, TIF assay etc) we analysed the DNA damage response by exposing DC cell lines to ionizing radiation. Our results demonstrated that DC cell lines have an abnormal DNA damage response and as a result show radiosensitivity. We have also knocked down the DKC1 gene in normal cell lines using siRNA oligonucleotides and demonstrated that this knock-down causes radiosensitivity. Therefore our results conclusively show an abnormal DNA damage response in cells derived from DC patients. Finally we used TA-65, a novel telomerase activator derived from the plant Astragalus membranaceus and showed radioprotective effects of this compound in normal lymphoblastoid cell lines. Taken together our results potentiate further the link between telomere maintenance and DNA damage response.

The role of Spr1851 protein Streptococcus pneumoniae in cell division Human extracellular pathogen S. pneumoniae encodes unique serin/threonine protein kinase of Eucaryotic type StkP and its cognate phosphatase PhpP. This kinase affects number of cellular processes including virulence, competence, cell division and cell wall synthesis by phosphorylating its substrates. Hypothetical protein Spr1851 named Jag was identified as a new StkP substrate in the membrane fraction by comparing the wild-type phosphoproteomes with StkP deleted strain. This protein consists of three domains and an interdomain region that contains T89 phosphorylation site. There is a Jag_N domain with an unknown function at the N-terminus. The C-terminus contains two domains - KH and R3H, which are highly conserved and their expected function is binding of nucleic acid. The main aim of this work is to explain the function of Jag protein, to determine the effect of individual domains on the phenotype and the localization of the protein and to determine the role of phosphorylation on T89. The results confirm that Jag protein could play a role in cell division or cell wall synthesis. Furthermore, the results indicate that the Jag_N domain is essential for the localization of the protein into the membrane, whereas the KH and R3H domains are...

Variation in pollen viability and the meiotic behaviour of sweet cherry Prunus avium L. cv. Lambert were studied. Pollen viability was estimated using aceto-carmine. Three types of pollen grain were observed; large, triangular; small, aborted; and intermediate. Only the first type were considered to have been viable at time of blossom harvest. No significant differences were detected between trees, between branches, or within branches, except for a North and South branch of one tree. Highly significant differences were detected among blossoms in a bud. The relative frequency of abnormal pollen was 42.26 per cent. Premeiotic budwood was stored at -5°C. Subsequent forcing at 10°, 15°, 20°, and 25°C to bring the buds to meiosis, and staining in alcoholic hydrochloric acid-carmine, revealed meiotic abnormalities at all temperatures except 15°C. Early meiotic division stages of additional material forced at this temperature were synchronous, but asynchrony developed after first metaphase. The first meiotic division was characterized by the frequent presence of a single pair of univalents, which appeared to arise from desynapsis of a bivalent in diplotene. Meiotic abnormalities associated with the univalents were observed at later division stages. The metaphase-anaphase division was typified by sequential disjunction of the bivalents, characteristic for sweet cherry meiosis. The relative frequency of abnormal first meiotic divisions was 45.49 per cent. / Land and Food Systems, Faculty of / Graduate

Cell duplication in Escherichia coli involves complex events, coordinated with chromosome replication. Because of the importance of chromosomes in perpetuating the normal cell cycle the initiation of their replication must be coordinated with cellular division. Following initiation, the cell must replicate and segregate its chromosomes, create a site necessary for septation and divide. These events could be coordinated by either; (1) biochemical reactions involving diffusible enzymes, or (2) multienzyme complexes which are localized at the site of DNA replication and cell division. In the latter case, the cyclic events of replication, segregation and cell division may be coordinated by physical-chemica1 or biochemical means. In any case, physical association implies pleiotropic effects. To test this hypothesis, cell division of the initiator mutant of E. coli , isolated by Kohiyama (1968) was studied. The temperature-sensitive initiator mutant E. coli CR 34T83 (ts DnaA) grew normally at 30 C, and at the restrictive temperature (42 C). The DNA replication as measured by radioactive precursor uptake, stopped after approximately 40 minutes and was equivalent to completion of rounds of replication started. Measurement of ribo- and deoxyribonucleotide triphosphate pools by thin-layer chromatography at 30 C and 42 C indicated residual DNA synthesis was not due to a limitation in the DNA precursors. Using a combination of density and differential radioactive labelling for the starts and ends of chromosomes, a preferred place for reinitiation of new replication cycles was shown. It was shown that DNA replication at 42 C terminated at a fixed region of the chromosome, and was identical to the 150 μg/ml chloramphenicol sensitive step involved in the process of initiation of chromosome replication in E. coli. A cessation of cellular division was noted by measurement of cell growth by Coulter Counter, at a shift from 30 C to 42 C, resulting in filamentous growth. Upon a return to 30 C, the cells resume division after approximately 15 - 20 min. The pleiotropic behaviour, that is, the cessation of cell division and initiation of DNA replication was a result of a point mutation in the gene DnaA, coding for a membrane bound protein involved in initiation. This mutation was mapped by transduction and was located at the isoleucine-valine region of the E. coli map. When this gene was transduced to different strains of E. coli K(12) the same pleiotropy was observed. This pleiotropy could be uncoupled, however, at 30 C by inhibitors of DNA synthesis or initiation. During recovery at 30 C from growth under 42 C, expression of cell division was proportional to cell equivalents generated at the restrictive temperature. RNA and protein synthesis, for 10 minutes during the recovery period, was obligatory for initiation of new rounds of replication, but not for the expression of cell division. A cell division "potential" protein was present under the restrictive growth condition. This "potential" was made at a derepressed rate and underwent a rapid degradation if kept at 42 C. At any given time, when returning from 42 C to 30 C, this "potential" allowed expression cell division based on DNA/mass or normal cell equivalents generated at 42 C. The half-life for decay of the division "potential" was estimated to be 1.4 minutes. The results were interpreted, in terms of an enzyme complex, which is common to the initiation of DNA replication and cellular division. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function of TONNEAU at the PPB?” To address this question, a series of constructs were generated to determine if TON2 binds calcium. Additionally, Ca2+ binding sites were mutated in constructs containing the TON2 gene fused to GFP or YPF. These constructs were then transformed into ton2 mutant plants and the localization of TON2 fusion protein and whether the construct is capable of rescuing the mutant phenotype were observed. Although, localization of TON2 to the PPB was not observed, the presence of the constructs were confirmed in the transformed plants using selection markers and by observing fluorescence under a confocal microscope.

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