Autosomal recessive primary microcephaly (MCPH, MIM 251200) is a neurodevelopmental disorder that results from a loss of neural progenitors in the embryonic neocortex. Patients with MCPH have a significantly small brain and exhibit reduced cognition. MCPH is a genetically heterogeneous disease involving mutations in thirteen genes, nine of which centrosome protein-coding genes, one of which is CDK5RAP2. The centrosome is the major microtubule organizing center in all animal cells. While MCPH is a neural stem cell disease, the molecular mechanisms for the disease remains unknown. Proteomic analysis of a mutant in the Drosophila CDK5RAP2 ortholog, centrosomin (cnn), we discovered proteins involved in intermediary metabolism, oxidative stress, and inherited Parkinson's disease that were post-translationally modified in mutant brains relative to wild type brains. These findings led us to discover that cnn mutants have neurological defects, including poor locomotor and flight performance, and are less active. We further demonstrated that cnn and Sas-4 (MCPH6/CPAP in human) mutant cells have elevated reactive oxygen species (ROS) levels, chronically activating the Jun N-terminal kinase (JNK) stress signaling pathway and thus activating FOXO by nuclear localization. The cause for these stress responses appears to be due to a severe deficiency in autophagy induction in MCPH mutant cells. Autophagy is a major catabolic pathway for the degradation of damaged proteins and organelles. Here we show that MCPH proteins are required for autophagy induction and act downstream of mechanistic target of rapamycin (mTOR) kinase, a negative regulator of autophagy. Together these results demonstrate a novel function for MCPH genes in oxidative stress and regulating autophagy. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / November 7, 2014. / autophagy, centrosome, microcephaly, oxidative stress / Includes bibliographical references. / Timothy Megraw, Professor Directing Dissertation; Yoichi Kato, Committee Member; Branko Stefanovic, Committee Member; Yanchang Wang, Committee Member.

Using the fruit fly Drosophila melanogaster as a model organism, this dissertation dissects molecular and biological functions of three proteins: Rootletin (Root), testis-specific Centrosomin (CnnT) and Spermitin (Sprn). Centrosomes are the major microtubule-organizing centers (MTOCs) in animal cells and each one consists of a pair of centrioles, a mother and a daughter centriole, surrounded by the pericentriolar material (PCM). Centrosomes play critical roles in cell division, cell polarization and intracellular trafficking, etc. In some cell types, the mother centriole matures to organize the primary cilium that is important for cell signaling and sensory perception. Cilia are linked at their base to the cell body by a cytoskeletal structure called the rootlet, whose detailed functions remain largely unknown. We have identified a protein called Rootletin (Root) that localizes at the rootlet in ciliated neurons. Root is essential for normal neuron-specific behaviors of the flies, including locomotion, mechanosensation, chemosensation and hearing. Furthermore, ultrastructure studies revealed that Root is required for organizing the rootlet, and we found that rootlet assembly is centriole-dependent. Altogether, we will define the important role of Root in rootlet organization and its requirement for ciliary functions. Mitochondria are energy centers in cells. In Drosophila, they also participate in sperm tail elongation by providing a structural platform for microtubule (MT) organization to support the elongating tail. Centrosomin (Cnn) has several variants, its centrosomal forms (CnnC) are essential for functional centrosomes. We discovered that the other non-centrosomal class of Cnn splice products (CnnT) is mainly expressed in fly testes. And unlike CnnC, which localizes at centrosomes, CnnT localizes to spermatid mitochondria (nebenkern). Cell culture and in vivo studies show that CnnT is necessary and sufficient to recruit MT-nucleating factors to nucleate MTs on mitochondria. Our study also suggested CnnT is required for normal sperm elongation and male fertility. Overall, we propose that CnnT promotes assembly of unique MTOCs on the surface of mitochondria where, in elongating sperm cells, it facilitates sperm tail growth. In the last chapter, we present our study on a novel testis-specific mitochondrial protein named Spermitin (Sprn). Using fluorescence microscopy, we found that Sprn is a mitochondrial protein localizing in the matrix of the spermatid nebenkern. However, Sprn is dispensable for normal sperm development and male fertility. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / October 30, 2014. / centrosome, cilia, mitochondrion, MTOC, rootlet, spermatogenesis / Includes bibliographical references. / Timothy L. Megraw, Professor Directing Dissertation; Branko Stefanovic, Committee Member; Yanchang Wang, Committee Member; Yi Zhou, Committee Member.

Plants are often subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates under abiotic stress conditions, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications are also required to facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent epigenetic pathway that regulates transcriptional silencing of loci across the genome. As critical regulators of gene expression, MOP1 and ABA pathways predictably regulate specific genes in a coordinated manner. In one project, the amino acid sequence of DNG103 and the gene promoter region were analyzed for conserved domains and cis-responsive elements, respectively. DNG103 is similar to the Arabidopsis ROS1, contains the conserved domains of a DNA glycosylase with DNA demethylase activity, and contains ABA-responsive elements (ABREs) in its promoter region. Transcript levels of Dng103, and the ABA-responsive gene Viviparous 1 (Vp1), were monitored in maturing embryos from two genotypes placed in culture under different conditions. Expression of both genes decreased after culture in hormone-free medium and was induced by ABA in Mop1 wildtype. Dng103 and Mop1 showed decreased expression in mop1-1 and dng103 mutants, respectively. Dng103 is not responsive to ABA in the mop1-1 mutant and Vp1 has reduced sensitivity. Therefore, DNG103, MOP1 and ABA might have common regulatory targets. Protoplast isolation and transfection protocols were standardized alongside the production of reporter constructs containing the Dng103 and Vp1 promoters. This technology will allow for the promoter characterization of genes of interest through quantifiable luciferase expression, using different conditions and genotypes. To identify genome-wide ABA-induced, MOP1-dependent and independent transcriptional responses, mop1-1 and Mop1 homozygous seedlings were subjected to exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes (DEGs) were identified in four pairwise comparisons. Overall, the loss of MOP1 exaggerated some ABA-induced changes in gene expression. The highest number of DEGs were identified in ABA-induced mop1-1 mutants, including many transcription factors. A gene regulatory network was used to predict relationships between DEGs; suggesting multifaceted regulatory scenarios including direct and indirect transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a hierarchical, cascading network of responsive genes. Additionally, a modest increase in CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in some genotypes, suggesting that MOP1 might be necessary to achieve environmentally induced transcriptional responses in maize. To understand the multistep ABA response of identified ABA-induced genes, an ABA-time course was carried out in another project using four different time points. The previously predicted ABA-responsive transcription factors Hb41 and Bzip4 showed an early induction to ABA and the late embryogenesis abundant Rab17 gene was induced during the transition from early to late response. Together, these results indicate that MOP1 and ABA act at multiple levels within complex, connected transcriptional networks to mediate tissue-specific growth and responses to some abiotic stresses. LIST OF SUPPLEMENTARY FILES 1. Significant differentially expressed genes in analysis groups I-VIII. 2. GO term enrichment in groups I, II, V and VI. 3. Arabidopsis and maize homologous transcription factors and target genes in ABA transcription factor hierarchical network and their corresponding gene expression. 4. Transcription factors and target genes in ABA transcription factor hierarchical network separated by transcriptional levels. 5. Group model parameters per gene. 6. Genome-wide siRNA changes in mop1-1 mutant. 7. TGS2 target genes. 8. Sequence Capture (SeqCap) DNA methylation ratios in all sequence contexts. 9. Promoter DNA methylation for Mop1 wildtype ABA-responsive DEGs. 10. MOP1-ABA targets with a loss of siRNA and DNA methylation at ABRE sites. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 6, 2019. / Abscisic acid (ABA), Epigenetics, Mediator of paramutation (Mop1), RNA-dependent DNA-Methylation (RdDM), siRNAs, Zea mays / Includes bibliographical references. / Karen M. McGinnis, Professor Directing Dissertation; Hong Li, University Representative; Henry W. Bass, Committee Member; Brian P. Chadwick, Committee Member; Jonathan H. Dennis, Committee Member.

Insect flight muscle (IFM) is the preferred model system for visualizing actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Electron tomography (ET) of fast-frozen, actively contracting Lethocerus IFM has recently resulted in a model for the weak to strong transition in the myosin crossbridges that produce force (Wu et al., 2010). These myosin molecules consist of a motor domain (MD), a lever arm and a coiled-coil rod domain that forms the filament backbone. The MD and lever arm region together constitute the subfragment 1 (S1) domain. The MD contains the ATP catalytic site and the actin-binding site. The myosin lever arm contains the essential and regulatory light chain bound to a long alpha helix. The first 50 nm of the rod domain consists of the subfragment 2 (S2) region, which acts both as a linker and adapter to transmit force produced by the MD and is sufficient to form the myosin dimer. Atomic models of myosin heads remodeled to fit the cross-bridge density show a distinctly straightened appearance when compared to the crystal structures of myosin subfragment 1 (S1). This implies that there is an aspect of the structural changes that occurs in force production that has not been recognized in myosin crystal structures of various intermediates. A weak-to-strong binding transition involving an azimuthal reorientation of the myosin MD on actin could explain this observation provided that myosin's α-helical coiled-coil S2 domain emerged from the thick filament backbone at a particular location. Previous studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages. Here we have used ET of IFM fibers in rigor, in which the filament lattice has been swollen in low ionic strength buffer, to view where S2 emerges from the thick filament backbone as a test of the weak to strong transition. The results show that the S2 origins of those rigor myosin heads bound to the target zone of active muscle originate from the same region of the thick filament as implied by the position of the S1/S2 junction observed in active muscle. This shows the myosin heads in clear agreement with the previously proposed weak to strong transition model. In order to visualize IFM by ET, crossbridge samples must be sectioned because they are otherwise too thick. Sample preparation methods include fixation and embedding followed by sectioning and staining. These preparation steps can potentially induce artifacts. The most notable sectioning artifacts are compression and shearing of the specimen. We examined ~80 nm thick transverse sections (cross-sections) cut with both a vibrating knife (sonic-knife) and static knife and explored different knife settings. We examined the mitigating effects of these sectioning parameters on both compression during cutting and shear distortions on the filaments as seen in tomograms of rigor muscle swollen in low ionic strength medium. Separate from specimen preparation challenges, data collection also presents with a set of artifacts. Mass loss in plastic sections in conventional ET can reduce section thickness by as much as 30%. We evaluated the benefit of collecting tilt series at -190°C with < 60 e⁻/Å2 total exposure, a value that is 50% of the dose typically used in cryo-ET for frozen hydrated specimen, in order to minimize radiation-induced mass loss that results in section thinning. Reducing the artifacts in our sample facilitated reconstruction of the IFM lattice making it possible to probe aspects of muscle contraction in greater detail than previously possible. ET is most useful for imaging biological structures in situ. A sample with reduced artifacts opens the door for better reconstructions. We used subvolume averages of both thin and thick filaments to reassemble the filament lattice with high signal-to-noise ratio averages. We used the improved samples with the intent of resolving the subfilament structures in the thick filament backbones as well as the subunit structure in the actin thin filaments. The information obtained from these averages provided two separate frames of reference for deciphering the relationship between crossbridge origins on the thick filament with crossbridge binding sites on actin. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / October 27, 2014. / Actin, Insect Flight Muscle, Lethocerus, Myosin, Thick filament, Thin filament / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; Timothy Logan, University Representative; P. Bryant Chase, Committee Member; Thomas C. S. Keller, Committee Member; Scott Stagg, Committee Member.

D4Z4 is a subtelomeric macrosatellite repeat on chromosome 4q that codes for DUX4, a gene that is causal to the muscle wasting disease Facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression is influenced by a number of genetic and epigenetic modifiers, including variation in D4Z4 copy number, single nucleotide polymorphisms (SNPs) on 4q, DNA and histone methylation changes, modifier genes like SMCHD1 and DNMT3B, and telomeres. The overarching goal of the research presented in this dissertation was to demonstrate the feasibility of targeting the disease locus using genome editing tools. To achieve this, we first needed to identify suitable cellular platforms for subsequent genome editing experiments. Through genotyping analysis, we were able to identify a cell line (HCT116) that was well-suited to studies investigating D4Z4/DUX4 expression, given that it harbors a disease-permissive 4qA allele. Using HCT116 and three of its DNA methyltransferase knockouts (1KO, 3BKO and DKO), we probed for factors that influence DUX4 expression in these cell lines. These experiments revealed that H3K9me3 loss and CpG hypomethylation can independently result in DUX4 expression in non-myogenic cell types. HCT116 and its DNMT KOs offer a new platform for studying DUX4 expression, albeit with some caveats. Importantly, we showed existence of D4Z4 transcripts in a variety of adult human tissues in addition to testis, with notably high expression in the thymus. Using these cell lines, we next explored the mechanism by which modifiers of D4Z4, such as SMCHD1 and telomeres might influence DUX4 expression. We generated several independent SMCHD1 knockout clones in HCT116 using TALENs. Characterization of these KOs revealed that despite no detectable changes in H3K9me3 or CpG methylation at D4Z4, SMCHD1 loss causes expression of unspliced DUX4, a phenomenon that was phenocopied by treatment of cells with a chemical inhibitor of telomerase. Spliced and pathogenic DUX4 was only expressed in these KOs upon treatment with 5-Aza-C, which demethylates DNA and lowers H3K9me3 levels at D4Z4. Given these results and the previously known importance of H3K9me3 enrichment in transcription and splicing, we speculate on a model, where SMCHD1 protein and telomeres, may act in coordination to provide an extra layer of transcriptional repression in addition to H3K9me3, at D4Z4. To achieve the main goal of this project, we took four independent approaches aimed at directly or indirectly repressing DUX4. In the first approach, we successfully deleted the array from a 4q permissive allele (in HCT116 and DKO cells), generating recombinants that likely had a shortened 4q chromosomal end and harbored an exogenously provided telomere seeding construct. Although we were unable to isolate clones of D4Z4-deleted cells, these results showed that the entire array can be deleted, also highlighting adverse side-effects of such targeting on cell viability. For our second approach, we used a CRISPR-based effector system to upregulate SMCHD1 transcript levels. Using a dCas9-VP64 activator construct, we were able to affect >2-fold upregulation in SMCHD1 in 293T cells. This avenue can be explored further to assess the effect of such upregulation on DUX4 and its target gene expression, in clinically relevant cell types. A SNP, resulting in a non-canonical polyadenylation (poly-A) sequence in DUX4 exon 3 is believed to stabilize pathogenic DUX4 transcripts in somatic cells. In a novel third approach, we designed pairs of gRNAs flanking the poly-A and deleted it using Cas9 nuclease recruited by these gRNAs. Significant lowering of DUX4 transcription in a mutant clone of HCT116, which contains a deletion of the pathogenic poly-A highlighted the importance of this sequence in transcript stabilization. Additionally, through lentiviral transduction of patient myoblasts, we showed that this approach can not only repress DUX4 expression, but also alleviate misregulation of a subset of its downstream target genes that are also biomarkers of FSHD. For our final approach, we enriched H3K9me3 at D4Z4 using a the dCas9-KRAB repressor system and a suitable gRNA. In both DKO cells and patient myoblasts, targeting repressed DUX4 expression significantly. Additionally, DUX4 target genes were also repressed in myoblasts and we showed that this repression was a consequence of increase and spread of H3K9me3 at D4Z4. Taken together, the results from these studies have generated promising future directions that can help understand the mechanism of how modifiers regulate D4Z4 expression. More importantly, by demonstrating the feasibility of targeting the disease locus in different cell types (including patient myoblasts), we have laid the foundation for development of future cell-based therapies to alleviate patient suffering in FSHD. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / March 7, 2018. / CRISPR, D4Z4, Epigenetics, FSHD, Genome editing, Macrosatellites / Includes bibliographical references. / Brian P. Chadwick, Professor Directing Dissertation; Michelle N. Arbeitman, University Representative; Thomas C. Keller, Committee Member; David M. Gilbert, Committee Member; Jonathan H. Dennis, Committee Member.

Human mesenchymal stem cells are a class of adult multipotent cells that are of interest to researchers for their clinical potential. While this cell type has been intensely investigated, there is still a significant amount to be learned about how these cells function. The presented studies utilized small molecular compounds to investigate the abilities of hMSCs. Project one investigated the roles matrix metalloproteinases (MMPs) play in adipogenic differentiation of hMSCs. MMPs are a family of metzincin proteinases that cleave the extracellular matrix (ECM), an activity that is critical to the differentiation process. These studies utilized the novel YHJ series of mercaptosulfonamide-based MMP inhibitors, which display high selectivity and potency for intermediate and deep pocket MMPs. Differentiation assays, kinetic assays, and qPCR were employed to characterize these inhibitors and determine their affect upon adipogenesis. Even though the YHJ MMPIs are not as stable as the hydroxamate-based MMPI GM6001, they produced comparable effects. Reduction in Lipid accumulation was also comparable to inhibition of peroxisome-proliferator activated receptor gamma (PPARγ) with the antagonist T0070907. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a potent agonist of PPARγ. This is important as MMP inhibition is a potential avenue for obesity treatment research. Finally, the selectivity provided by the YHJ MMPIs allowed for the identification of MMP-3 (stromelysin-1) as a possible regulator of adipogenesis. Project two investigated hMSC resistance to the apoptotic effects of chemotherapeutic drugs. This has been of major interest, as hMSCs can confer resistance to tumor microenvironments. However, the effects of internalized chemotherapeutics upon hMSCs remain largely unexplored. Cellular viability and proliferation assays, combined with different biochemical approaches, were used to investigate the effects of Paclitaxel exposure upon hMSCs. The results indicate that hMSCs are highly resistant to the cytotoxic effects of Paclitaxel treatment, even though there was no detectable expression of the efflux pump P-glycoprotein, the usual means by which a cell resists Paclitaxel treatment. Moreover, Paclitaxel treatment induces hMSCs to adopt a non-proliferative fibroblastic state, as evidenced by changes to morphology, cellular markers, and a reduction in differentiation potential that is not directly coupled to the cytoskeletal effects of Paclitaxel. Taken together, these results show that Paclitaxel treatment does not induce apoptosis in hMSCs, but does induce quiescence and phenotypic changes. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2015. / June 29, 2015. / Adipogenesis, Drug Resistance, Matrix Metalloproteinases, Mesenchymal Stem Cells, MMP Inhibitors, Paclitaxel / Includes bibliographical references. / Qing-Xiang Sang, Professor Directing Dissertation; Gregory Dudley, University Representative; Michael Roper, Committee Member; Timothy M. Logan, Committee Member; Yan Li, Committee Member.

Extracellular vesicles (EVs) include exosomes and microvesicles, and are important mediators of cell-to-cell communication in healthy and pathological environments. Packed with biological information and present in a variety of biological fluids, EVs offer exciting promise for biomarker discovery and applications in therapeutics and non-invasive diagnostics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis, cargo packaging, and secretion remain unknown. To an extent, these findings have been restricted by the limited cells from which vesicles have been characterized utilizing the same enrichment method. The following work contributes to advancement of knowledge surrounding EV formation within the cell, protein trafficking into EVs, and the content and functions of oncogenic vesicle secretion in cancer. In Chapter 2 of this dissertation, vesicle secretion is characterized across sixty cancer cell lines from the National Cancer Institute (NCI-60) by nanoparticle tracking analysis. The quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Positive correlates of exosomal-sized vesicle secretion included Rab GTPases and mediators of sphingolipid metabolism, while larger microvesicle-sized vesicle secretion was associated with gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. To investigate the global protein content of NCI-60 cell derived EVs, pure vesicle isolates were harvested for a large-scale mass spectrometry approach, detailed in Chapter 3. This work provides the largest proteomic profile of EVs in a single study, identifying 6,071 proteins with 213 common to all isolates. Proteins included established EV markers, and vesicular trafficking proteins such as Rab GTPases and tetraspanins. The identification of differentially-expressed proteins may offer future candidates for earlier cancer diagnosis and prognostic monitoring of disease. Network analysis of vesicle quantity and proteomes identified EV components associated with vesicle secretion, including CD63, CD81, syntenin-1, VAMP3, Rab GTPases, and integrins. Finally, integration of vesicle proteomes with whole-cell molecular profiles revealed similarities, suggesting EVs provide a reliable reflection of their progenitor cell content, and are therefore excellent indicators of disease. Together, these observations reveal new insights into key players involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. In both the aforementioned works, a tetraspanin protein, CD63 was identified and confirmed for its role in small vesicle biogenesis. CD63 is a conserved protein enriched in late endosomal and lysosomal compartments and has been widely used as an exosome marker. However, little is understood about the mechanisms of CD63-mediated exosome formation, or its interacting proteins. Accumulating evidence has suggested that an Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1) may interact with CD63 within the exosomal pathway. Viruses share similar sizes and structures as exosomes, and recent evidence suggests that viruses can hijack the exosome pathway to modulate cell-to-cell signaling. Indeed, LMP1 trafficking into multivesicular bodies (MVBs) can alter the content and function of exosomes. LMP1-modified exosomes can in turn enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. In efforts to advance knowledge surrounding the role of CD63 in exosome production and vesicular protein sorting, original work in Chapter 4 demonstrates the importance of CD63 presence for exosomal packaging of LMP1. In this study, a role of LMP1 in vesicle production that requires CD63 is described, providing an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Further evidence revealed the role of CD63 in limiting LMP1-induced non-canonical NF-κB and ERK activation. Overall, these findings have implications in future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in the context of viral- or nonviral- associated tumorigenesis. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy. / Spring Semester 2017. / February 17, 2017. / Biomarkers, Cancer, Epstein-Barr virus, Exosomes, Extracellular vesicles, Proteomics / Includes bibliographical references. / David G. Meckes, Jr., Professor Directing Dissertation; Scott M. Stagg, University Representative; Pradeep G. Bhide, Committee Member; Myra M. Hurt, Committee Member; James M. Olcese, Committee Member.

The follicular epithelium (FE) of the Drosophila egg chamber is an excellent model system to study cell-cycle regulation, cell differentiation and cell migration in development. During oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle (stage 1-6), endocycle (stage 7-10a), and gene amplification (stage 10b-13). Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation and inactivation in follicle cells are essential for the mitotic cycle/endocycle (M/E) and the endocycle/gene amplification (E/A) switches, respectively. In my dissertation, I mainly focus on Notch signaling and its downstream target broad (br). In the first part of the dissertation, I introduce the background information of the egg chamber system, Notch signaling and other associated factors. In the second part, I describe a screen strategy to identify novel genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. In the third part, I select a Notch target gene br from the above-mentioned screen and study its regulation and functions. I will show br, encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. My findings also suggest an important role of br in the timing of follicle cell development during the M/E switch. In the fourth part, I report the uniform pattern of Br in the follicular epithelium is gradually lost in the anterior follicle cells (stretched cells and border cells) from stage 9 to 10a during oogenesis. This downregulation of Br is functionally significant for proper stretched-cell stretching. I also find ecdysone and JAK/STAT signaling mediate the downregulation of Notch-maintained Br. Together, My research investigates the complex Notch signaling network, and reveal that Notch-directly-regulated Br interacts with the ecdysone and JAK/STAT pathways, serving as an important spatiotemporal cue for proper cell differentiation and morphogenetic movement during Drosophila oogenesis. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the Doctor of Philosophy. / Spring Semester, 2015. / February 27, 2015. / broad, ecdysone, follicle cell, JAK/STAT, Notch, oogenesis / Includes bibliographical references. / Wu-Min Deng, Professor Directing Dissertation; Jamila I. Horabin, University Representative; Jonathan Hancock Dennis, Committee Member; Timothy Megraw, Committee Member; Steven John Lenhert, Committee Member.

The Spindle Assembly Checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects. Bipolar kinetochore attachment is required for SAC silencing which allows for anaphase onset and the release of the phosphatase, Cdc14. The Cdc14 early anaphase release (FEAR) pathway reverses Cdk1 activity to promote early anaphase events. Using S. cerevisiae, we discovered that the FEAR pathway inhibits SAC activity in anaphase, a process we have named SAC termination. We found that hyper-activation of FEAR allows premature SAC silencing, which depends on Cdc14-mediated dephosphorylation of a kinetochore protein Fin1, a regulator of protein phosphatase PP1. Surprisingly, in fin1∆ mutants we found dynamic kinetochore localization of SAC protein Bub1 after anaphase entry, indicating Fin1 regulates SAC localization. Fin1-PP1 promotes SAC termination through the dephosphorylation of the outer kinetochore Ndc80 as well as the removal of Ipl1 kinase. These results show that FEAR activation during early anaphase promotes SAC termination through Fin1-PP1 to ensure no SAC activation during anaphase. We also discovered that S-phase cyclin dependent kinase (Clb5/CDK) promotes the correction of erroneous attachments to promote kinetochore biorientation. We show that clb5Δ mutants are sensitive to syntelic attachments due to prolonged SAC arrest. This phenotype was partially dependent on Cnn1, as cnn1Δ partially rescued clb5Δ sensitivity to syntelic attachments. This result suggests that Clb5/Cdk1 inhibits Cnn1 during S-phase/early mitosis to promote bipolar establishment. Finally, we revealed that a group of inner kinetochore proteins, the Constitutive Centromere Associated Network (CCAN), prevents SAC silencing in the presence of tensionless attachments. The kinetochore protein Ybp2 interacts with the CCAN and we found that ybp2Δ partially suppresses the SAC silencing mutant ipl1-321, indicating that Ybp2 functions to promote SAC silencing. Surprisingly, ybp2Δ fully rescues CCAN mutant sensitivity to syntelic attachments indicating that the CCAN inhibits Ybp2 to prevent SAC silencing. Interestingly, ybp2Δ mutant cells show enhanced phosphorylation of a kinetochore protein Dsn1 further supporting the hypothesis that Ybp2 antagonizes Ipl1 kinase to promote SAC silencing. In summary, our results uncover new molecular mechanisms that suppress SAC activity which allows for efficient segregation of the genome. We uncover that the FEAR pathway promotes SAC termination by decreasing kinase activity while simultaneously increasing phosphatase activity on the kinetochore during anaphase. This ensures no SAC activity during anaphase. Additionally, we uncover that S-phase Cdk1 promotes the correction of erroneous kinetochore-microtubule attachments by inhibiting Cnn1 kinetochore function. This allows for efficient error correction before anaphase onset. Lastly, we found that a network of inner kinetochore protein complexes, CCAN, prevents SAC silencing when tensionless attachments are present, revealing how the CCAN supports accurate chromosome segregation. Taken together, the data presented here deepens our understanding of SAC regulation which ensures faithful chromosome segregation. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / July 11, 2018. / CCAN, Cdc14, Fin1, PP1, S-phase Cdk, Spindle Assembly Checkpoint / Includes bibliographical references. / Yanchang Wang, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Akash Gunjan, Committee Member; Timothy Megraw, Committee Member.

Apparatus has been developed for the irradiation of V79-379A Chinese hamster lung fibroblast cells with 3.6 MeV protons from the Van de Graaff accelerator at the National Accelerator Centre in Faure. The original intention of this work was to investigate and measure a possible dose rate effect on the survival of V79 cells, in the dose range from zero to 25 Gy, at dose rates of about 3 Gy/s and 300 Gy/s. The survival curves initially obtained were anomalous in that they showed abnormally high levels of survival and a tendency to remain at a constant survival level for doses above 10 Gy. Systematic attempts to correct this observed anomaly, involved the following; apparatus improvements were made, a means of measuring the beam profile was devised, the current measuring device and the dosimetry were improved and a possible dose rate effect on intracellular oxygen was investigated. After these improvements, the anomalous effect was much reduced, but not entirely eliminated. The final results showed no significant difference between the survival of cells irradiated at dose rates of about 3 Gy/s and 300 Gy/s; qualitative differences were however noticeable. After correction for the effect of a non-uniform beam profile, the survival curves were significantly different to published work. This difference suggested a possible dose rate effect between dose rates of about 0.1 Gy/s and dose rates above 3 Gy/s.