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<p>Centromeres are essential chromosomal loci at which kinetochore formation occurs for spindle fiber attachment during mitosis and meiosis, guiding proper segregation of chromosomes. In humans, centromeres are located at large arrays of alpha satellite DNA, contributing to but not defining centromere function. The histone variant CENP-A assembles at alpha satellite DNA, epigenetically defining the centromere. CENP-A containing chromatin exists as an essential domain composed of blocks of CENP-A nucleosomes interspersed with blocks of H3 nucleosomes, and is surrounded by pericentromeric heterochromatin. In order to maintain genomic stability, the CENP-A domain is propagated epigenetically over each cell division; disruption of propagation is associated with chromosome instabilities such as aneuploidy, found in birth defects and in cancer. </p><p>The CENP-A chromatin domain occupies 30-45% of the alpha satellite array, varying in genomic distance according to the underlying array size. However, the molecular mechanisms that control assembly and organization of CENP-A chromatin within its genomic context remain unclear. The domain may shift, expand, or contract, as CENP-A is loaded and dispersed each cell cycle. We hypothesized that in order to maintain genome stability, the centromere is inherited as static chromatin domains, maintaining size and position within the pericentric heterochromatin. Utilizing stretched chromatin fibers, I found that CENP-A chromatin is limited to a sub-region of the alpha satellite array that is fixed in size and location through the cell cycle and across populations. </p><p>The average amount of CENP-A at human centromeres is largely consistent, implying that the variation in size of CENP-A domains reflects variations in the number of CENP-A subdomains and/or the density of CENP-A nucleosomes. Multi-color nascent protein labeling experiments were utilized to examine the distribution and incorporation of distinct pools of CENP-A over several cell cycles. I found that in each cell cycle there is independent CENP-A distribution, occurring equally between sister centromeres across all chromosomes, in similar quantities. Furthermore, centromere inheritance is achieved through specific placement of CENP-A, following an oscillating pattern that fixes the location and size of the CENP-A domain. These results suggest that spatial and temporal dynamics of CENP-A are important for maintaining centromere and genome stability.</p> / Dissertation

<p>Cnidarians are often considered simple animals, but the more than 13,000 estimated species (e.g., corals, hydroids and jellyfish) of the early diverging phylum exhibit a broad diversity of forms, functions and behaviors, some of which are demonstrably complex. In particular, cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Some cubozoan species possess complex mating behaviors or particularly potent stings, and all possess well-developed light sensation involving image-forming eyes. Like all cnidarians, cubozoans have specialized subcellular structures called nematocysts that are used in prey capture and defense. The objective of this study is to contribute to the development of the box jellyfish Alatina alata as a model cnidarian. This cubozoan species offers numerous advantages for investigating morphological and molecular traits underlying complex processes and coordinated behavior in free-living medusozoans (i.e., jellyfish), and more broadly throughout Metazoa. First, I provide an overview of Cnidaria with an emphasis on the current understanding of genes and proteins implicated in complex biological processes in a few select cnidarians. Second, to further develop resources for A. alata, I provide a formal redescription of this cubozoan and establish a neotype specimen voucher, which serve to stabilize the taxonomy of the species. Third, I generate the first functionally annotated transcriptome of adult and larval A. alata tissue and apply preliminary differential expression analyses to identify candidate genes implicated broadly in biological processes related to prey capture and defense, vision and the phototransduction pathway and sexual reproduction and gametogenesis. Fourth, to better understand venom diversity and mechanisms controlling venom synthesis in A. alata, I use bioinformatics to investigate gene candidates with dual roles in venom and digestion, and review the biology of prey capture and digestion in cubozoans. The morphological and molecular resources presented herein contribute to understanding the evolution of cubozoan characteristics and serve to facilitate further research on this emerging cubozoan model.

<p> Dinoflagellates possess large genomes in which most genes are present in many copies. This has made studies of their genomic organization and phylogenetics challenging. Recent advances in sequencing technology have made deep sequencing of dinoflagellate transcriptomes feasible. This dissertation investigates the genomic organization of dinoflagellates to better understand the challenges of assembling dinoflagellate transcriptomic and genomic data from short read sequencing methods, and develops new techniques that utilize deep sequencing data to identify orthologous genes across a diverse set of taxa. To better understand the genomic organization of dinoflagellates, a genomic cosmid clone of the tandemly repeated gene Alchohol Dehydrogenase (AHD) was sequenced and analyzed. The organization of this clone was found to be counter to prevailing hypotheses of genomic organization in dinoflagellates. Further, a new non-canonical splicing motif was described that could greatly improve the automated modeling and annotation of genomic data. A custom phylogenetic marker discovery pipeline, incorporating methods that leverage the statistical power of large data sets was written. A case study on Stramenopiles was undertaken to test the utility in resolving relationships between known groups as well as the phylogenetic affinity of seven unknown taxa. The pipeline generated a set of 373 genes useful as phylogenetic markers that successfully resolved relationships among the major groups of Stramenopiles, and placed all unknown taxa on the tree with strong bootstrap support. This pipeline was then used to discover 668 genes useful as phylogenetic markers in dinoflagellates. Phylogenetic analysis of 58 dinoflagellates, using this set of markers, produced a phylogeny with good support of all branches. The <i>Suessiales</i> were found to be sister to the <i>Peridinales.</i> The <i>Prorocentrales </i> formed a monophyletic group with the Dinophysiales that was sister to the <i>Gonyaulacales.</i> The <i>Gymnodinales</i> was found to be paraphyletic, forming three monophyletic groups. While this pipeline was used to find phylogenetic markers, it will likely also be useful for finding orthologs of interest for other purposes, for the discovery of horizontally transferred genes, and for the separation of sequences in metagenomic data sets.</p>

Integrin signaling is critical for many biological functions including cell survival, cell migration, development, immunity and wound healing. Integrins perform their function through a structural change that is propagated from the cytoplasm to the ligand binding domain in inside-out signaling and from the ligand binding domain to the cytoplasm during outside-in signaling events. However, the structural basis for the signal transduction in a native-like lipid bilayer environment is poorly understood. We investigated the inactive and active conformations of integrin alpha IIb beta 3 in a membrane environment to understand the structural basis of integrin signaling. We used reconstituted small unilamellar vesicles to mimic the native membrane environment and used cryo-electron tomography of ice embedded specimens and 3-D averaging to obtain the structures. Our results showed that, in this membrane environment, both active and inactive integrins are in an upright conformation. They differ in the separation of the leg regions of the alpha and beta chains. Inactive integrins have the legs together, similar to the 3-D structure of detergent solubilized alpha IIb beta 3 observed in ice but with a more upright orientation with respect to the membrane. The active integrins have the legs separated by about 5.6 nm at the membrane surface. These results support a model in which integrin signaling is achieved by the relative movement of the leg regions of the two subunits. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Fall Semester, 2005. / Date of Defense: November 10, 2005. / Cryoelectron Tomography, Conformational Change, Integrin / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; Michael S. Chapman, Outside Committee Member; Thomas M. Roberts, Committee Member; Thomas C. S. Keller III, Committee Member; Kenneth H. Roux, Committee Member.

A seqüência nxh1 foi isolada a partir de uma biblioteca de cDNA da glândula de veneno da cobra coral Micrurus corallinus. Essa sequência apresenta similaridade estrutural com as toxinas de \"três dígitos\", e principalmente com as &#945;-neurotoxinas, devendo apresentar 4 pontes dissulfeto deduzidas por comparação com outras proteínas desta família. Porém, essa potencial toxina não possui alguns aminoácidos importantes para a interação com o receptor nicotínico de acetilcolina na junção neuromuscular. A potencial toxina NXH1 foi expressa em E. coli de 3 maneiras distintas. Os melhores resultados foram obtidos quando a NXH1 foi expressa em fusão com uma cauda de histidina. Essa construção permitiu uma rápida e eficiente purificação da proteína recombinante. A proteína de fusão foi usada para a produção de um soro específico anti-NXH1. O soro anti-proteína recombinante, assim como o soro do Instituto Butantan, reconheceu a toxina recombinante em Western blot e ELISA. Além disso, o anti-NXH1 reconheceu em Western blot apenas uma banda presente no veneno de M. corallinus, mas não reconheceu os venenos de outras 10 espécies de Micrurus. Experimentos de ligação mostraram que componentes do veneno de M. corallinus ligam-se aos receptores nicotínicos das membranas de músculo de rato. O soro anti-NXH1 não inibiu a ligação do veneno aos receptores, indicando que, ou a NXH1 não é uma &#945;-neurotoxina, ou o antisoro não consegue impedir a ligação da toxina nativa ao receptor, ou que existem outras a-neurotoxinas do veneno que não são bloqueadas pelo soro anti-NXH1. / The nxh1 sequence has been isolated from a cDNA library from the coral snake Micrurus corallinus\' venom gland. The deduced protein is highly similar to known \"three finger\" &#945;-neurotoxins, with four deduced disulfide bridges. However, the predicted protein lacks some important amino acids for the nicotinic acetylcholine receptor interaction. The potencial toxin NXH1 was expressed in E. coli in three different ways. The best results were obtained when the protein was expressed as a His-tagged fusion protein, which allowed rapid and efficient purification of the recombinant toxin. The fusion protein was used to generate a specific antiserum against NXH1. The produced antiserum, as well as the serum from Instituto Butantan, recognized in ELISA and Western blot the recombinant toxin. Besides, the anti-NXH1 serum recognized in Western Blot a single band from the venom of M. corallines but not the venom from other 10 Micrurus species. Binding experiments showed that the components from M. corallines venom bind to nicotinic acetylcholine receptor in rat muscle membranes. The anti-NXH1 serum did not inhibited the binding of the venom to the receptors. It seems that NXH1 is not an &#945;-neurotoxin, or that the antiserum does not inhibit the binding of the native toxin to the receptor, or that the antiserum is not capable to inhibit the action of other &#945;-neurotoxin from the venom.

ORF45 of Kaposi's sarcoma–associated herpesvirus (KSHV) is a gamma herpesvirus-specific, immediate-early, and tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other co-purified proteins, including prominent ones of ~38 kDa and ~130 kDa. Mass spectrometry revealed that the 38 kDa protein is viral ORF33 and the 130 kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl terminal 19-aa of ORF45, and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7, in both transfected conditions and KSHV-infected cells. Moreover, we noticed an ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation, which was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. To determine if the binding of ORF33 is a critical function of C19, we used co-precipitation with point mutants of the C19 region and identified two required residues: tryptophan 403 and tryptophan 405. We then engineered KSHV genomes containing these mutants and transfected them into iSLK cells. Similar to the C19 deletion mutant, we found that both binding-deficient mutants exhibited decreased ORF33 accumulation and viral particle production. Since C19 is sufficient for binding ORF33, we hypothesized that introduction of a C19 analogue could inhibit binding and may lead to a similar decrease in viral particle production. We used ELISA to measure the binding of ORF33 to ORF45 in the presence of TAT-C19 and found that TAT-C19 inhibited binding in a dose-dependent manner. To measure the analogue's effect on viral particle production, we treated KSHV-infected cells with TAT-C19 and found that TAT-C19 inhibited viral particle production in a dose-dependent manner. In conclusion, binding of ORF33 and ORF45 during the lytic cycle is required for accumulation of the ORF33 and production of viral particles. In addition to forming a complex with ORF33 and USP7, we also found a strong association of ORF45 to RSK and ERK during the lytic cycle, matching previous reports that ORF45 bound ERK and RSK in a single complex. During that study, ORF45 was found to form a complex with ERK and RSK and the formation of this complex lead to accumulation of active ERK and RSK. While the binding site of RSK was mapped to aa 55-70, it was unclear if ERK bound to ORF45 directly or through RSK. Using in vitro binding analysis, we identified an ERK binding site in aa 16-35. Using T-Coffee analysis, we compared the sequences of gamma-2 herpesvirus homologues of ORF45 and found two highly conserved phenylalanine residues at aa 32 and 34. After generating point mutants of each residue to alanine, we measured their effect on the activation of ERK and RSK induced by ORF45 and found that either mutation lead to decreased activation of ERK and RSK. We are currently evaluating their effect upon the activation of ERK and RSK during the lytic cycle and the production of progeny virions. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the Doctor of Philosophy. / Fall Semester 2015. / November 3, 2015. / ERK1/2, Kaposi's sarcoma-associated herpesvirus, KSHV, ORF33, ORF45, p90RSK / Includes bibliographical references. / Fanxiu Zhu, Professor Directing Dissertation; Scott Stagg, University Representative; Hengli Tang, Committee Member; Thomas C. S. Keller, III, Committee Member; Kathyrn M. Jones, Committee Member.

Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on KSHV progeny virion production. Importantly, we investigated the regulation of gene expression by ORF45-actvated RSK by performing RNA-seq of KSHV-infected cells. We show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. One of the few viral substrates of ORF45-activated identified by our mass spectrometry analysis was ORF36. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. We report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in their binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N-terminus of ORF36 or an acidic patch of ORF45 abolished the binding. Additionally, dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we employed BAC mutagenesis to engineer both ORF36-null and kinase-dead (KD) mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45, and indicate a significant role of ORF36 in the production of infectious progeny virions. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates viral and cellular kinase signaling to optimize lytic replication. The findings reported here have important implications for the pathobiology of KSHV and other diseases in which RSK activity is missregulated. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2016. / March 22, 2016. / Kaposi's sarcoma, kinase, KSHV, lytic replication, ORF45, RSK / Includes bibliographical references. / Fanxiu Zhu, Professor Directing Dissertation; Qing-Xiang Sang, University Representative; Hengli Tang, Committee Member; David Gilbert, Committee Member; Jonathan H. Dennis, Committee Member.

DNA replication in all Eukaryotes follows a defined temporal order termed replication-timing program (RT), which is coupled with the spatial separation of chromatin distribution inside the nucleus. Early or late replicating chromatin self-organizes in 3D into sub-nuclear compartments at the nucleus interior or proximity to nuclear lamina respectively. RT is also highly correlated with multiple other features of the genome including transcriptional activity, chromatin composition, and mutational landscape. The molecular mechanisms regulating RT and linking these events are unclear. To investigate the role of DNA sequences in RT regulation, I adopted two parallel approaches to test the sufficiency and necessity of specific DNA segments in these processes. In the first approach, I developed an extra-chromosomal vector system (E-BAC) to show that determinants for RT and A/B compartmentalization are genetically encoded in ~200kb DNA sequences. In the second approach involving CRISPR (clustered regularly interspaced short palindromic repeats) mediated genome-editing, I identified three “early replication control elements” (ERCEs) internal of the domain that act redundantly and interdependently to give rise to both early replication and A/B compartmentalization of a pluripotency associated domain in mouse embryonic stem cells. The three ERCEs and other ERCE-like elements form the strongest CTCF-independent interactions among each other, which could drive the formation of A/B compartments inside the nucleus. The ERCEs also display a combination of active chromatin features resembling promoters and/or enhancers. They are implicated in gene regulation possibly by mediating the formation of transcription factories. These findings underscore the genetic influence on controlling multiple cellular processes, and highlight the complexity of cis regulation from the linear genome. The discovery of cis regulatory elements offers mechanistic insight linking highly correlated genomic features/activities, and provides opportunities to further dissect their relationship from a 3D perspective. Deeper understanding of genome regulation will hopefully enable the manipulation of these processes in cell function and disease. / 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. / April 4, 2018. / Chromatin compartment, Chromatin interactions, cis-elements, DNA replication timing, E-BACs, ERCEs / Includes bibliographical references. / David M. Gilbert, Professor Directing Dissertation; Akash Gunjan, University Representative; Jonathan H. Dennis, Committee Member; Fanxiu Zhu, Committee Member; Debra A. Fadool, Committee Member.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of significant public health concern closely related to other highly pathogenic flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). With the rise of ZIKV in Brazil in 2015, its potential link to microcephaly and other severe neurological birth defects prompted the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. Since this time, numerous studies have provided ample evidence to establish ZIKV as the causative agent of microcephaly, yet the molecular mechanisms underlying these neurodevelopmental defects are not well understood. We therefore establish a tractable experimental model system to investigate the impact of ZIKV on human neural development. We demonstrate that ZIKV efficiently infects human cortical neural progenitor cells (hNPCs) derived from induced pluripotent stem cells, but less efficiently infects other cells along the neural differentiation pathway, including immature cortical neurons. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection disrupts cell cycle progression and induces cell death in hNPCs contributing to their attenuated growth. Global transcriptome analyses of ZIKV-infected hNPCs reveal transcriptional dysregulation, notably a downregulation of cell-cycle-related genes, highlighting the potential involvement of cell cycle pathways in ZIKV biology. We then study the molecular mechanisms by which ZIKV manipulates the cell cycle in hNPCs and the functional consequences of cell-cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that host cell-cycle disruption is unique to ZIKV among the flaviviruses tested, including DENV and WNV, however similar among the two strains of ZIKV tested, including the prototype Uganda strain and a Puerto Rican strain. ZIKV, but not DENV, infection induces DNA double-strand breaks, triggering the DNA damage response through the ATM/Chk2 signaling pathway, while suppressing activation of the ATR/Chk1 signaling pathway in hNPCs. Furthermore, ZIKV infection impedes the progression of cells through S phase thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with S-phase inhibitors leads to an increase in ZIKV replication, but not of WNV or DENV replication. Together, our results identify hNPCs as a direct target of ZIKV and the damaging impact of ZIKV on the growth of hNPCs. Importantly, our data demonstrate ZIKV’s ability to induce host DNA damage and arrest cell cycle progression, which results in a cellular environment favorable for its replication. As hNPCs generate the cortical neurons during early fetal brain development, the ZIKV-mediated growth retardation likely contributes to the neurodevelopmental defects of the congenital Zika syndrome. / 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. / January 23, 2018. / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Timothy Megraw, University Representative; Brian P. Chadwick, Committee Member; David M. Gilbert, Committee Member; Yan Li, Committee Member; Fanxiu Zhu, Committee Member.