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.

Zika virus is an arbovirus that has reemerged in recent years as a global health concern. A member of the Flavivirus genus, Zika virus is closely related to other major human pathogens such as dengue virus and West Nile virus, but each of these viruses exhibit varied clinical pathologies. While Zika virus was first discovered in 1947, it was not until the outbreaks across the Pacific and Americas from 2013 onward that Zika virus’ unique neuropathies were revealed. Of particular concern was the potential for vertical transmission and teratogenic effects in infected pregnant women, prompting the World Health Organization to declare Zika virus a public health crisis of global concern in 2016. Even with a growing knowledge of Zika virus’ adverse fetal outcomes and the characterization of congenital Zika syndrome, much research is still needed to understand the cellular and molecular mechanisms underlying these potentially devastating infection consequences. Given this need, we therefore established a cerebral organoid system to model Zika virus exposure throughout fetal brain development. When exposed to Zika virus, organoids, which recapitulated features of brain development in the first trimester, exhibited microcephaly-like restrictions in maturation. This included an overall reduction of organoid growth, enlargement of the ventricle-like structures, and increased cell death in organoids exposed to both the prototypical and modern epidemic strains of Zika virus. In older organoids resembling the fetal brain structure of the second trimester, Zika virus preferentially infected neural progenitor cells as compared to neurons, and also infected several other early brain cell types, demonstrating the potential cellular affects underlying Zika virus-induced microcephaly. We next utilized a neuroglia-derived cell line highly permissive to Zika virus infection to find Zika virus host factors in neural cells. We identified the host protein, AXL, as an important factor for Zika virus infection, but observed a difference in the requirement of AXL for the infection of Zika and dengue virus. Chimeras of Zika and dengue virus revealed that the structural proteins displayed on the virion surface were a major determinant of AXL-dependent viral infection. Lastly, we investigated the molecular mechanisms contributing to AXL-dependent infection and found that suppression of the IFN response as mediated by AXL was minimally involved in AXL-dependent Zika virus infection, in contrast to dengue virus, suggesting that different aspects of AXL signaling may facilitate Zika and dengue infection in neuroglia. Our data provide a model system for evaluating Zika virus exposure during fetal brain development. We show that Zika virus infects both neural progenitor cells and neuroglia, cell types which are present at different stages of cerebral cortex development. Our work highlights the importance of AXL in Zika virus infection of neuroglia and the differential requirement of AXL for Zika and dengue virus infection. Collectively, these findings contribute toward dissecting the mechanism by which Zika virus utilizes AXL for viral entry and infection, which may be important for understanding Zika virus pathogenesis as AXL is expressed on many of the cell types infected by the virus in vivo. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / October 28, 2019. / AXL, dengue virus, organoid, Zika virus / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Timothy Megraw, University Representative; Jonathan Dennis, Committee Member; Yan Li, Committee Member; Fanxiu Zhu, Committee Member.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation.  To better understand the transmission genetics of hop we genotyped 4,512 worldwide accessions of hop, including cultivars, landraces, and over 100 wild accessions, using a genotyping-by-sequencing (GBS) approach.  From the resulting ~1.2M single nucleotide polymorphisms, pre-qualified GBS markers were validated by inferences in population structures and phylogeny. Analysis of pseudo-testcross mapping data from F1 families revealed mixed patterns of Mendelian and non-Mendelian segregation. We used genome-wide association studies and FST analysis to demonstrate selection mapping of genetic loci for key traits, including sex, bitter acids, and drought tolerance. Among the possible mechanisms underlying the observed segregation distortion from the genomic data analysis, the cytogenetic analysis points to meiotic chromosome behavior as one of the contributing factors. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase.  To explore the role of  meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI. Our initial DAPI survey of late meiotic prophase nuclei in two cultivars and two wild hop revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including autosomes. We then systematically examined all stages of meiosis for meiotic irregularities utilizing telomere and 5S rDNA fluorescent in-situ hybridization (FISH). We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree.  To date, linkage groups in hop have not been assigned to physical chromosomes, rendering genome assemblies and karyotype development particularly challenging, given its genomic structural variability. Consequently, understanding genome evolution in Humulus represents a considerable challenge, requiring additional resources, including integrated genome maps. In order to facilitate cytogenetic investigations into the transmission genetics of hop, we report here the identification and characterization of 18 new and distinct tandem repeat sequence families. A tandem repeat discovery pipeline was developed using k-mer and dot plot analysis of PacBio long-read sequences from the hop cultivar, Apollo.  We produced oligonucleotide FISH probes for some of these families and demonstrated their utility via 3D FISH to stain meiotic chromosomes from wild hop, var. neomexicanus. Probes derived from the tandem repeat sequence families, HSR0, HuluTR120 and HuluTR235, were shown to be particularly useful for detection of meiotic abnormalities in wild and cultivated hop. Collectively, these tandem repeat sequence families represent unique and valuable new reagents with the capacity to inform genome assembly efforts and support comparative genomic analyses. The findings shed light on long-standing questions on the unusual transmission genetics and phenotypic variation in hop, with major implications for breeding, cultivation, and the natural history of Humulus. The data in this dissertation have been or are being considered for publication. The data presented in Chapter 2 has been published in The Plant Genome (Zhang et al., 2017). The data presented in Chapter 2 has been published in Frontiers in Plant Science (Easterling et al., 2018). The data in Chapter 4 is submitted to Frontiers in Plant Science. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / October 30, 2019. / cannabaceae, chromosome, cytogenomics, humulus, meiosis / Includes bibliographical references. / Hank W. Bass, Professor Directing Dissertation; Cathy W. Levenson, University Representative; Austin R. Mast, Committee Member; Karen M. McGinnis, Committee Member; Brian G. Miller, Committee Member.

The evolutionary arms race for survival against phages and other mobile genetic elements has produced a wide repertoire of defensive strategies in prokaryotes. CRISPR (Clustered, regularly-interspaced, short palindromic repeats) systems are adaptive immune pathways that use RNA-guided nucleases to target and destroy foreign nucleic acid. The ease with which these CRISPR associated (Cas) enzymes can be reprogrammed to target almost any gene sequence has revolutionized biological research, industry, and biomedical applications. Human genome editing with CRISPR-Cas is ongoing and has immense therapeutic potential to correct disease causing mutations at the DNA level but is plagued by off-target cleavage events and inefficient activity for certain targets. Extensive structural and biochemical studies on CRISPR-Cas9 have provided critical understanding of the molecular mechanisms that govern enzymatic activity, but many questions still need to be answered for Cas9 to become an effective and safe tool. In this work, I investigate the structural parameters of Cas9 responsible for specificity and efficiency using directed evolution and in vitro characterizations on a thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). I identified a surprising role of the phosphate lock for tuning specificity in a manner that depends on residue size and charge. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to off-targets. An increase in the size of the substituted residues further reduces the sensitivity to guide-DNA mismatches. I successfully engineered a catalytically enhanced AceCas9 with up to 4-fold increase in catalytic efficiency by targeting conformational states. These engineered variants provide a path forward to utilizing the slower type II-C Cas9s in genome editing applications as well as improving the activity of currently used genome editing Cas9. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / August 14, 2019. / CRISPR-Cas9, Directed Evolution, Efficiency, Protein Engineering, Specificity / Includes bibliographical references. / Hong Li, Professor Directing Dissertation; David Gilbert, University Representative; M. Elizabeth Stroupe, Committee Member; Hengli Tang, Committee Member; Qian Yin, Committee Member.

The Drosophila ovary is an important model system for studying oogenesis and has provided insight into broader, biological topics such as stem cell niche, differentiation, migration, morphogenesis, signaling, cell-size regulation, and tumorigenesis. The purpose of this dissertation was to build a comprehensive single-cell transcriptomic atlas of cells regulating oogenesis and ovulation and to use the ovarian follicle cells, as a model system to interrogate the role of ectopic Notch expression in development and during tumorigenesis. Single-cell RNA sequencing analysis revealed transcriptional signatures for each of the 28 expected ovarian cell types and developmental stages. Each stage of oogenesis was then separately analyzed with a special focus on the most diverse cell type, the follicle cells. This identified key cell-type specific expression patterns governing processes like differentiation, mitotic-to-endocycle switch, migration, morphogenesis, phagocytic removal of nurse cells, eggshell formation, and a newly identified shift of the pre-corpus lutuem cell expression from oogenesis-to-ovulation. Additional characterization of the interconnected tissues in the dataset identified novel cellular heterogeneity of the oviduct and a population of hemocytes associating closely with it. We further describe how this association occurs in a non-mating-dependent manner as early as the pupal stage (during oviduct development). Additional characterization of the hemocyte cluster reveals expression of many phagocytic genes including the newly identified enzymatic marker, Cp1. Additional validation of cell-type markers classifies these macrophage-like cells as plasmatocytes, one of the most common types of hemocytes in flies. Armed with a rich dataset describing proper signaling during development we turn to the follicle cells and switch focus to study dysregulated signaling of the Notch pathway and its involvement in tumor formation. First, we expressed ectopic Notch (NICD) in follicle cells and discovered an uneven pattern of nuclear NICD retention in a cell-cycle dependent manner. We find that the cell-cycle regulator, string (cdc25), can strongly impact the localization of NICD regardless of the cell-cycle status of the cell. We also identify that Vps proteins involved in forming the ESCRT complex can similarly regulate the nuclear NICD pattern. Next, we examined the role of ectopic Notch signaling in tumorigenesis and found that while not sufficient for tumor formation, Notch functions as a tumor promoter and leads to a more dysplastic tumor phenotype. Tumor cells with ectopic NICD gain a survival advantage which may be due to a bypass of the DNA damage sensing checkpoint. RNA sequencing revealed unique expression of these tumor cells with ectopic NICD in both pre- and post-tumor conditions. Specifically, we identified DNA stability related genes, RecQ4 and Xpd, which are upregulated in NICD-overexpressing pre-tumor cells. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 8, 2019. / Drosophila, hemocyte, Notch, oogenesis, ovulation, tumor / Includes bibliographical references. / Wu-Min Deng, Professor Co-Directing Dissertation; Kathryn Jones, Professor Co-Directing Dissertation; Timothy Megraw, University Representative; Kimberly Hughes, Committee Member; Steven Lenhert, Committee Member.

This dissertation focuses on microtubule assembly mechanisms and functions in dividing and non-dividing cells. In dividing cells, centrosome is the major microtubule-organizing center (MTOC). Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome, an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here, we characterize the single Nin ortholog in Drosophila. Drosophila Nin localizes to the periphery of the centrosome, but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian ortholog of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of dividing stem cells. Surprisingly, loss of nin expression from a nin mutant does not significantly impact embryonic and brain development, fertility, or locomotor performance of mutant flies, nor their survival upon exposure to DNA damaging agents. While not essential, Nin localizes to non-centrosomal MTOCs (ncMTOCs) in wing epithelia and muscle, two types of specialized and differentiated cell types, suggesting that Nin plays a supportive role in non-centrosomal microtubule organization. ncMTOCs have a variety of roles presumed to serve the diverse functions of the range of non-dividing cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in another differentiated Drosophila cell type, fat body cells. This perinuclear ncMTOC in fat body cells is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Nin to assemble non-centrosomal MTs and does so independently of the widespread MT nucleation factor -tubulin. Patronin does not antagonize with known MT depolymerases in fat body ncMTOC, but acts cooperatively with Ninein to assemble circumferential MTs, and also recruit MT polymerase Msps to promote elongation of radial MTs. Functionally, the fat body ncMTOC is essential for retrograde dynein-dependent endosomal trafficking to restrict plasma membrane growth and for the secretion of basement membrane proteins. Together, we identify an ncMTOC with novel architecture and MT regulation properties that serves a vital secretory function. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 1, 2019. / centrosome, microtubule-organizing center, Msps/XMAP215, Ninein, non-centrosomal MTOC, Patronin/CAMSAP / Includes bibliographical references. / Timothy L. Megraw, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Yanchang Wang, Committee Member; Robert J. Tomko, Jr., Committee Member.

Specific mutations in the replacement histone variant H3.3 are known to drive cancers such as glioblastomas, chondroblastomas and large cell tumors of the bone primarily in children and young adults. Several recent studies have suggested that transcriptional defects associated with H3.3 mutations are likely to be involved in tumor formation. However, transcription independent roles of H3.3 and their potential contribution to cancer have not been investigated. Here we report that histone H3.3 (but not the related replication dependent H3.1 or H3.2 variants) is recruited within ~60 seconds to sites of laser induced DNA damage. This recruitment is dependent on acetylation at the N terminus of the protein. A stable depletion of this protein in human cells leads to high levels of endogenous DNA damage and an impaired ability to repair this damage, leading to genomic instability and sensitivity to DNA damaging agents. Flies with reduced levels of H3.3 are also sensitive to damaging agents and accumulate spontaneous damage. H3.3 depletion alters the recruitment of double strand break (DSB) repair factors, with defects in the recruitment of proteins involved in homologous recombination (HR) repair and faster recruitment of some proteins involved in non-homologous end joining (NHEJ). We also find that the cancer associated mutants are defective in localizing to damage sites and their expression leads to an accumulation of high levels of endogenous damage. Overall, our data from evolutionarily distant species suggest that histone H3.3 plays a crucial role in HR-mediated DNA repair which is conserved across eukaryotes. Hence, based on the strong links between defective DNA repair and cancer, we propose that the DNA repair defects associated with H3.3 mutations are likely to contribute to genomic instability, and thereby to carcinogenesis, independent of the known transcriptional roles of histone H3.3. Based on these findings, we propose a potential therapeutic strategy that combines the use of inhibitors along with a DSB inducing agent to selectively target cells that are defective in HR repair due to H3.3 depletion or mutation. Our promising preliminary data show that this is effective in H3.3 knockdown cells and may also be useful in killing H3.3 cancer mutants. This knowledge helps us to understand the complex details of these deadly mutations and hopefully brings us closer to a treatment. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy. / Fall Semester 2015. / October 29, 2015. / cancer, chromatin, DNA repair, H3.3, histone / Includes bibliographical references. / Akash Gunjan, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Yanchang Wang, Committee Member; Jonathan Dennis, Committee Member.

abstract: Arachnids belong to the phylum Arthropoda, the largest phylum in the animal kingdom. Ticks are blood-feeding arachnids that vector numerous pathogens of significant medical and veterinary importance, while scorpions have become a common concern in urban desert cities due to the high level of toxicity in their venom. To date, viruses associated with arachnids have been under sampled and understudied. Here viral metagenomics was used to explore the diversity of viruses present in ticks and scorpions. American dog ticks (Dermacentor variabilis) and blacklegged ticks (Ixodes scapularis) were collected in Pennsylvania while one hairy scorpion (Hadrurus arizonensis) and four bark scorpions (Centruroides sculpturatus) were collected in Phoenix. Novel viral genomes described here belong to the families Polyomaviridae, Anelloviridae, Genomoviridae, and a newly proposed family, Arthropolviridae. Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals. Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations. / Dissertation/Thesis / Masters Thesis Microbiology 2019

No description available.

No description available.