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To exit a non-proliferative state and enter cell division, metazoan cells require external signals to facilitate activation and metabolic reprogramming. As cell growth is required before cell division, cells redirect their metabolism for de novo synthesis of cell building blocks, including phospholipids for cell membrane construction. How cells coordinate initial signaling events with metabolism is unknown. Lineage-specific factors transmit activating signals via cell surface receptor-ligand interactions. Among these are PI3K/AKT, MAPK/ERK, and JAK/STAT, all of which have been described to contribute to metabolic regulation. In particular, the signal transducer and activator of transcription (STAT) is a transcription factor with broad roles in cell cycle progression and glucose metabolism. Previous data from our laboratory found that one STAT family member, STAT3, was one of the primary signaling pathways activated when transitioning out of a resting state. Inhibition of STAT3 was found to suppress the initiation of cell growth and citrate levels, a main intermediate for fatty acid synthesis, suggesting a connection to cell metabolism. This thesis investigates the role of STAT3 in the regulation of metabolism in cells transitioning from a resting state to a cell growth state. The first chapter of this thesis provides relevant background information on the metabolic and signaling pathways involved in a resting and cell growth state. It also provides data that supports an important role for STAT3 during initial cell growth. The second chapter demonstrates the importance of STAT3 in multiple cell types using a small molecule inhibitor of STAT3, STAT3 knockdown, and knockout experiments. I also establish a potential link between STAT3 and the metabolic enzyme citrate synthase (CS) for the synthesis of citrate. In the third chapter I show that STAT3 transcriptionally regulates CS through two binding sites, CS1 and CS2. Finally, I determine that CS is essential for initial cell growth and that exogenous citrate can rescue the loss in cell growth and proliferation observed in the CS and STAT3 knockdown cells. Together, these findings describe a novel mechanism for initial cell growth whereby signaling and metabolic events are tightly linked to regulate the transition from a resting state to a state of initial cell growth. These results may uncover new strategies to block the initiation of proliferation in human pathological conditions including tumor recurrence and autoimmunity. / Graduate

Cytokinesis separates replicated chromosomes and cytoplasm into two daughter cells. In animal cells, this is achieved by the formation of a cleavage furrow that bisects the mitotic (or meiotic) spindle. It is known that the mitotic apparatus defines the cell cleavage plane. However, it is not clear how the mitotic apparatus initiates the cleavage furrow. Each part of the mitotic apparatus; namely asters, central spindle (microtubule arrays and the spindle midzone), and chromosomes, has been found capable of inducing a cleavage furrow in certain cell types. Yet it is uncertain which part is the essential source of the signal and whether all parts act in concert. This thesis systematically examines in grasshopper spermatocytes 1) which spindle constituent is the essential source of furrow signal; 2) the impact of microtubules on distribution of actin filaments and positioning of cell cleavage relative to spindle reorganization; 3) the independent role of the spindle midzone relative to microtubules in furrow initiation and ingression. These examinations combine micromanipulation with digital-enhanced polarization microscopy and epifluorescence microscopy, in which mitotic spindles in living cells are mechanically dissected and rearranged as desired as well as microfixed to evaluate and propose models for cell cleavage. This thesis has come to the conclusion that none of structural constituents of the spindle apparatus is essential for cell cleavage induction except microtubules. First, furrow induction occurs regardless of a particular spindle constituent, so long as sufficient microtubules are present to form bipolar arrays. Second, microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage. Asymmetric alterations of spindle microtubules dynamically affect the location of the spindle midzone, distribution of actin filaments, and ultimately position of the cleavage furrow in cells containing a bipolar spindle, monopolar spindle, or half-spindle. Third, actin filaments are distributed to the furrow region by microtubule-mediated transport, but organized by the midzone, which is essential for furrow ingression, but not initiation. These results suggest that during post-anaphase spindle assembly, actin filaments are excluded by bipolar microtubule arrays to the equatorial cell cortex where they bundle into a contractile ring with cytokinetic factors. / Graduation date: 2004

The longstanding view has been that metabolism allows for cell division to take place, but that metabolic processes do not actively promote cell division. I have recently challenged this notion by identifying a unique gain-of-function metabolic mutant in the budding yeast Saccharomyces cerevisiae. Moderate over-expression of Abf2p, a conserved mitochondrial DNA (mtDNA) maintenance protein, increases the amount of mtDNA by 100-150%. I have shown that cells moderately over-expressing Abf2p can out-proliferate their wild type (WT) counterparts, initiate DNA replication sooner, and increase in size faster than WT cells. Yeast grown under certain conditions in continuous cultures become synchronized with respect to their oxygen consumption, displaying distinctive oxidative and reductive phases. In cells over-expressing Abf2p, the reductive phase is expanded compared to that of WT cells. Since glutathione, the cell?s main redox buffer and sulfur containing metabolite, peaks during this phase, I asked if sulfur metabolism was altered in cells with more mtDNA. Sulfur metabolite levels are increased ~40% in cells over-expressing Abf2p. Furthermore, exogenous addition of various sulfur containing compounds, which is known to increase sulfur metabolic flux, caused WT cells to increase in size faster and initiate DNA replication sooner, mimicking the phenotype seen in cells moderately overexpressing Abf2p. I then investigated possible interactions between sulfur metabolism enzymes and the yeast Cdk, Cdc28p. Performing co-immunoprecipitation experiments, two enzymes of the sulfur metabolic pathway were found to bind Cdc28p. One of these, Cys4p, lies at the critical junction point between the pathways leading to the formation of glutathione versus one carbon metabolism. The interaction of the enzymes with Cdc28p appears to be dependent on progression through the cell cycle, and preliminary evidence suggests that Cdc28p/Cys4p binding may peak at the G1/S transition of the cell cycle. In summary, I have identified a unique gain-of-function metabolic mutant in S. cerevisiae that leads to accelerated initiation of DNA replication. Sulfur metabolic flux is up-regulated in cells over-expressing Abf2p, and exogenous sulfur sources added to WT cultures phenocopied cells over-expressing Abf2p. Most importantly, I have shown a physical interaction between sulfur metabolic enzymes and the Cdk driving the cell cycle in yeast.

Thesis (M.D.S.)--University of Adelaide, 1985. / Some ill. mounted. Includes bibliographical references (leaves 84-95).

Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998. / Errata is pasted onto back end paper. Includes bibliographical references (26 leaves).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The meiotic behaviour and pollen fertility of Lambert sweet cherry (Prunus avium L.) and irradiation propagates of Lambert were investigated. Chromosome clumping, due to associations between the centromere region of non-homologous chromosomes, characterized early meiotic stages in Lambert. While the associations, which were evident as chromatic centers, complicated interpretation of pachynema, the pachytene morphology of the eight chromosomes was described. Except for small chromatic areas delimiting the centromere, all chromosomes were basically achromatic. Three chromosomes had an additional one or two chromatic regions, which in all cases were in the short arm. One chromosome, designated number 3, had a major heterozygous interstitial deletion in the short arm. Marked repulsion between the homologous chromosomes during diplonema resulted in a diffuse stage. The single pair of univalents, occasionally observed throughout meiosis and considered to be of desynaptic origin, were first detected in diplonema. Subsequent meiotic stages were usually normal, and the incidence of abnormal meiosis and tetrad stages containing micronuclei were estimated to be 9-62 per cent and 3.24 per cent respectively. Meiosis was characterized by synchronous division within the anther locules. Cytoplasmic connections were evident between neighbouring meiocytes during meiosis I, and cytomictic phenomena were observed occasionally. Abortion of 50 per cent of the developing microspores occurred prior to the first mitotic division of the spore nucleus, and was attributed to the heterozygous deletion in chromosome 3. Pollen abortion at anthesis was estimated to be about 42 per cent. Annual or regional effects on the incidence of abnormal meiosis or pollen abortion were not evident. Forcing of blossom buds at 15°C did not appear to affect meiotic behaviour. Twelve irradiation propagates of Lambert were investigated. Of these, seven had pollen fertility similar to the parental Lambert clone, and their meiotic behaviour also was comparable. The remaining five propagates had reduced pollen fertility. One propagate was found to have a greatly increased incidence of the single pair of univalents, as compared to parental Lambert; two propagates contained a reciprocal translocation; and two contained a paracentric inversion. The extent of pollen abortion was predictable from the meiotic behaviour in only one of the latter five propagates. Pollen studies revealed significant annual variation among the 12 irradiation propagates, some varying more than others. With one exception, forcing of blossom buds at 15°C did not appear to affect meiotic behaviour. / Land and Food Systems, Faculty of / Graduate

The Min system ensures that gram-negative bacteria undergo symmetric cell division. The three Min proteins, MinC, MinD, and MinE, display a dynamic pattern of subcellular organization on the inner cell membrane that directs division proteins to the mid-cell. This process is driven by the ATPase activity of MinD that is stimulated through its interaction s with Min E. A recent structure of MinE in complex with MinD suggests that MinE undergoes a dramatic conformational change to allow MinD - binding residues to be released from the MinE hydrophobic core. However, this structure used a MinE mutant designed to favor this conformational change, raising questions regarding the mechanism by which wild - type MinE can undergo this transition in vivo. One potential scenario that might explain this structural change involves a recently discovered interaction between MinE and the membrane surface. To investigate the possibility that lipid binding could induce this structural transition in MinE, circular dichroism and enzyme kinetics studies were carried out. These studies were also done on MinE mutants designed to either eliminate membrane binding or induce the conformational change involved in MinD - binding. The results demonstrated that a membrane induced conformational change does occur, and requires the presence of a key lipid - binding region at the N - terminus. However, removal of this sequence failed to alter the kinetics of MinE - stimulated MinD - catalyzed ATP hydrolysis. Overall, our results provide a step forward in our understanding of the role of the interaction between MinE and the membrane in the Min system, but also highlight the need for additional investigation before this system might be used as a novel antibiotic target for pathogenic, gram - negative bacteria such as Neisseria gonorrhoeae.