The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development. Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our  findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues. / Master of Science

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No description available.

<p>Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. </p> <p>Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. </p> <p>Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. </p> <p>To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish <em>in vitro</em> biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). </p> <p>Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from <em>Pseudomonas aeruginosa</em> can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.</p>

<p>The rapid localized cell death at and around sites of attempted pathogen infection, termed the hypersensitive response (HR), is an immune response mechanism commonly utilized in plants. This cell death limits pathogens from accessing host nutrients which often leads to resistance. The interaction of pathogen signals and host receptors that are required for the HR are well studied; however, the processes that regulate cell death during the HR remain enigmatic. The plant lesion mimic mutants, which form spontaneous lesions and/or undergo autoactive cell death in the absence of infection or stress, are commonly used as model systems to study the HR. Some lesion mimic mutants are caused by autoactive alleles of the resistance genes that recognize pathogen signals and trigger the HR. These mutants have facilitated studies of the HR as they allow the study of the HR without the need to control for pathogen infection. Currently, the etiologies of most maize lesion mimic mutants are unknown. Lesion mimic mutants contain numerous metabolic perturbations, including the increased accumulation of salicylic acid (SA), phenylalanine, and intermediates in heme and chlorophyll biosynthesis and catabolism. Some of these perturbations are dependent on the cause of lesion formation. As such, the accumulation of any of these metabolites in a lesion mutant may infer the etiology of that mutant. This dissertation contains three projects related to the molecular characterization of HR and maize lesion mimic mutants. In the first project (Chapter 2), I compared the metabolite profile of 23 maize lesion mimic mutants. This work identified two major findings that were further explored in the other projects in this dissertation. The first major finding is that four of the 23 mutants have metabolic perturbations that are like those of the known HR lesion mutant, <em>Rp1-D21</em>. In project two (Chapter 3), I molecularly characterize, <em>Lesion10</em>, which is one of the mutants that has HR-like metabolic perturbations. Using genome-wide association studies, I identified a gene candidate that may modify <em>Lesion10</em> phenotypic severity. The second major finding from project one is that SA accumulates to higher than wild-type levels in most of the lesion mutants analyzed. In the third project (Chapter 4), I characterized how SA is synthesized in maize and if SA is necessary or sufficient for the formation of lesions during the HR in maize. Using untargeted metabolite analysis, stable isotope feedings, and enzyme assays, I provide evidence of both known SA biosynthetic pathways in maize and demonstrate that the two pathways are interdependent. In addition, I show that increased accumulation of SA is not required for the HR in maize.</p>

<p>Regulators of G protein signaling (RGS) proteins are key negative regulators of Gα signaling, a branch of G-protein-coupled receptor (GPCR)-mediated signal transduction. Approximately 35% of drugs approved by the Food and Drug Administration (FDA) target GPCRs, so it is not surprising that the discovery of RGS proteins has triggered an interest in them as new drug targets. Even though many studies have been shown the involvement of RGS proteins in cancers, there is still a knowledge gap in understanding function and regulation of RGS proteins in these diseases. Consequently, in this thesis, I explored roles of two RGS proteins that have been implicated in cancers.</p> <p>RGS2 is proposed to act as a tumor suppressor in many different cancers, such as breast cancer, bladder, and ovarian cancer. Here, we investigated if RGS2 also plays a tumor suppressor role in UM, whose growth is driven by overactivated Gαq/11 signaling. We found that increased expression levels of RGS2 inhibit cell growth of UM 92.1 and Mel-202 cells. Mechanistically, this cell growth inhibition is dependent on the association between RGS2 and Gαq, but independent of its canonical GTPase-accelerating protein (GAP) activity. Furthermore, RGS2 inhibited the Mitogen-activated protein kinases (MAPK) signaling, downstream of Gαq, while leaving Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activation unaffected. These data indicate a tumor suppressor role for RGS2 in UM and proposes RGS2 stabilization as a potential therapeutic targeting strategy. </p> <p>In contrast to RGS2, RGS20 contributes to cancer progression, particularly in breast cancer. However, how RGS20 is regulated is understudied. Palmitoylation, a reversible post-translational modification, regulates functions of other RGS proteins, and RGS20 is predicted to be palmitoylated. We provided direct evidence of RGS20 palmitoylation in cells and validated the palmitoylation site as the conserved cysteine (Cys148) in the RGS domain. Our results showed that palmitoylation on this site does not affect its GAP activity and subcellular localization, but it affects the association between RGS20 and active Gαo, and inhibition of Gαo-mediated signaling. This study serves as a foundation for future studies in furthering understating the role of palmitoylation in RGS20 function and its possible implications in cancer biology. </p>

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No description available.

There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.

Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.