Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 194-227).

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 85-95.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 123-149.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 110-123).

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 104-116.

This work addresses the mechanism of cell cycle specific actin reorganization in Drosophila syncytial blastoderm embryos. During mitosis in typical animal cells after chromosome segregation is complete, daughter cells are separated in a process called cytokinesis. Cytokinesis is ordinarily driven by constriction of an actin ring that physically pinches the cell in two. The early Drosophila embryo is a syncytium; nuclei divide in a single cell without intervening cytokinesis. During the later syncytial divisions, nuclei are arranged in a monolayer at the cortex of the embryo. This stage of embryogenesis is characterized by cycles of actin reorganization that are coordinated with the nuclear division cycles. Since several components of typical cleavage furrows function in this cell cycle driven actin reorganization, the syncytial blastoderm has been used as a model system to better understand cell cycle driven actin reorganization in typical cells. The syncytial Drosophila embryo is easily manipulated genetically, cytoskeletal structures can be visualized in both fixed and living embryos, and large quantities of embryos are attainable for biochemical analysis. We have therefore chosen this model system to study actin reorganization. We show that actin reorganization in syncytial embryos is coordinated by cell cycle cues similar to those utilized in typical cells. Drosophila embryo actin reorganization has several unique features, however. For instance, actin reorganization appears to be associated with centrosomes in a process that does not require microtubules. In addition, the driving force for formation of Drosophila cleavage structures may be actin filament polymerization, rather than contraction of an acto-myosin ring. Whether these characteristics of Drosophila embryo actin reorganization typifies actin reorganization in other cells remains to be seen.

Circadian rhythms are self-sustained 24-hour period oscillations present in most organisms, from bacteria to human. They can be synchronized to external cues, thus allowing organisms to anticipate environmental variations and optimize their performance in nature. In Drosophila, the molecular pacemaker consists of two interlocked transcriptional feedback loops. CLOCK/CYCLE (CLK/CYC) sits in the center and drives rhythmic transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). PER and TIM negatively feedback on CLK/CYC transcriptional activity, forming one loop, while VRI and PDP1 form the other by regulating Clk transcription negatively and positively, respectively. Posttranscriptional and posttranslational regulations also contribute to circadian rhythms. Although much has been learned about these feedback loops, we are still far from understanding how stable 24-hour period rhythms are generated. My thesis work was to determine by which molecular mechanisms kayak-α (kay-α) and Ataxin-2 (Atx2) regulate Drosophila circadian behavior. Both genes are required for the precision of circadian rhythms since knocking down either gene in circadian pacemaker neurons results in long period phenotype. The work on kay-α constitutes the first half of my thesis. We found that the transcription factor KAY-α can bind to VRI and inhibit VRI’s repression on the Clk promoter. Interestingly, KAY-α can also repress CLK’s transcriptional activity on its target genes (e.g., per and tim). Therefore, KAY-α is proposed to bring precision and stability to the molecular pacemaker by regulating both transcriptional loops. The second half of my thesis focuses on ATX2, an RNA binding protein whose mammalian homolog has been implicated in neurodegenerative diseases. We found that ATX2 is required for PER accumulation in circadian pacemaker neurons. It forms a complex with TWENTY-FOUR (TYF)—a crucial activator of PER translation—and promotes TYF’s interaction with Poly(A)-binding protein. This work reveals the role of ATX2 in the control of circadian rhythms as an activator of PER translation, in contrast to its well-established role as a repressor of translation. It also further demonstrates the importance of translational regulation on circadian rhythms. Finally, it may help understanding how ATX2 causes neuronal degeneration in human diseases.

Autophagy is a cellular process that delivers cytoplasmic materials for degradation by the lysosomes. Autophagy-related (Atg) genes were identified in yeast genetic screens for vehicle formation under stress conditions, and Atg genes are conserved from yeast to human. When cells or animals are under stress, autophagy is induced and Atg8 (LC3 in mammal) is activated by E1 activating enzyme Atg7. Atg8-containing membranes form and surround cargos, close and mature to become the autophagosomes. Autophagosomes fuse with lysosomes, and cargos are degraded by lysosomal enzymes to sustain cell viability. Therefore, autophagy is most frequently considered to function in cell survival. Whether the Atg gene regulatory pathway that was defined in yeast is utilized for all autophagy in animals, as well as if autophagy could function in a cell death scenario, are less understood. The Drosophila larval digestive tissues, such as the midgut of the intestine and the salivary gland, are no longer required for the adult animal and are degraded during the pupal stage of development. Cells stop growing at the end of larval development, and proper cell growth arrest is required for midgut degradation. Ectopic activation of the PI3K/Akt signaling induces cell growth and inhibits autophagy and midgut degradation. Down regulating PI3K/Akt pathway by Pten mis-expression activates autophagy. In addition, mis-expression of autophagy initiator Atg1 inhibits cell growth and knocking down autophagy restore PI3K/Akt activity. Together, these results indicate that autophagy and growth signaling mutually inhibit each other. Midgut destruction relies on the autophagy gene Atg18, but not caspase activation. The intestine length shortens and the cells undergo programmed cell size reduction, a phenomenon that also requires Atg18, before cell death occurs during midgut destruction. To further investigate whether cell size reduction is cell autonomous and requires other Atg genes, we reduced the function of Atg genes in cell clones using either gene mutations or RNAi knockdowns. Indeed, many Atg genes, including Atg8, are required for autophagy and cell size reduction in a cell autonomous manner. Surprisingly, Atg7 is not required for midgut cell size reduction and autophagy even though this gene is essential for stress-induced autophagy. Therefore, we screened for known E1 enzymes that may function in the midgut, and discovered that Uba1 is required for autophagy, size reduction and clearance of mitochondria. Uba1 does not enzymatically substitute for Atg7, and Ubiquitin phenocopies Uba1, suggesting Uba1 functions through ubiquitination of unidentified molecule(s) to regulate autophagy. In conclusion, this thesis describes: First, autophagy participates in midgut degradation and cell death. Second it reveals a previously un-defined role of Uba1 in autophagy regulation. Third it shows that the Atg genes are not functionally conserved and the requirement of some Atg genes can be context dependent.