<p> TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not &Delta;Np63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with &Delta;Np63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPAR&gamma; expression and consequently leading to a drastic decrease of the PPAR&gamma; target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.</p>

<p>Soybean (Glycine max) yield is significantly affected by soybean cyst nematode (SCN), Heterodera glycines, and causes an annual loss of billions of US dollars. In this study, Glycine max xyloglucan endotransglycosylase/hydrolase gene (Gm-XTH52) was transformed into a nematode susceptible G. max [Williams 82/PI 518671] variety of soybean to test whether the protein expression has a role in resistance to H. glycines, and possible chemical changes the expression may cause in the plant composition. Expression level of the Gm-XTH52 gene was three times higher than in controls. Significant reduction in the number of SCN cysts suggested suppression of H. glycines parasitism upon transformation. While total sugar amounts did not significantly differ between the transformed and control plants, xyloglucan amounts of loosely bound sugars of genetically mosaic plants were significantly lower in comparison to controls. Control plants showed lower molecular weight sugars than the transformed plants not subjected to H. glycines infection.

<p> Genome sequencing technology has enabled the identification of genetic variants that are linked with cancer phenotypes, whether these are somatically acquired mutations or common inherited single nucleotide polymorphisms (SNPs). Whereas coding variants have been reported to disrupt protein function to promote cancer, most variants map to noncoding regions, with no known function. Recently, much effort has gone into annotating the human noncoding genome, enabling the characterization of the functional basis of noncoding SNPs. As an example of functional impact, breast cancer (BrCa) risk-associated SNPs can alter transcription factor binding at distal enhancers. </p><p> Identifying the targets of risk SNPs remains a challenge. One reason for this is the complex three-dimensional structure of the genome. Local chromatin openness correlates with chromatin activity, and sites of chromatin that are open concurrently across multiple cell types indicates a functional relationship between them. We mapped BrCa risk-associated SNPs to regions of open chromatin to predict the most likely functional risk SNPs. Then, we predicted their targets by identifying the gene promoters whose openness correlated with these risk regions. Further, we validated a gene which is a novel therapeutic target and relevant in breast cancer biology. </p><p> In addition to SNPs, noncoding somatic mutations are also predicted to play a role in cancer. In 2012, driver mutations were reported in the telomerase gene promoter, hinting at the relevance of mutations in regulatory elements. This is particularly true when considering oncogenes whose elevated expression in certain cancers is not attributable to coding mutations or copy number amplification. We reveal the enrichment and functional nature of somatic mutations mapping to enhancers that regulate the estrogen receptor gene, which is known to drive over two-thirds of breast cancer. </p><p> Attributing function to noncoding SNPs and mutations associated with cancer risk and progression is a growing necessity in this era of whole-genome cancer biology. This thesis demonstrates a methodology to identify the functional consequence and gene targets of significantly mutated or risk variant-bearing enhancer sets to narrow the gap between known and unknown risk factors in BrCa.</p>

<p> Human breast cancer is currently the highest diagnosed form of cancer and the second leading cause of cancer-related deaths in American women. Triple negative breast cancer is of the basal subtype and displays the worst prognosis owing to its highly metastatic properties. Current treatments focused on eradicating breast tumors in lieu of or following local therapy include chemotherapy, hormonal therapy, and targeted therapy. Hormonal therapy is not an option for triple negative breast cancer as it does not contain hormone receptors and there are currently no approved biological targeted therapies. Chemotherapy has proven unsuccessful because triple negative breast cancer is highly drug resistant. Here we report that metastatic human breast cancer cells (BCCs) were converted to a less aggressive phenotype and overcame chemotherapeutic drug resistance following exposure to embryonic stem cells (ESCs) encapsulated in alginate microstrands. We also demonstrate that the 3D ESC model restores proper EGFR and canonical Wnt/&beta;-catenin signaling pathway regulation in metastatic BCCs and can be applied to identify a biological treatment for triple negative breast cancer. This study establishes the feasibility of inhibiting highly aggressive human BCCs with 3D cultured ESCs as demonstrated through decreases in metastatic BCC proliferation, abnormal metabolism, migration, invasiveness, chemotherapeutic drug resistance, and survival <i>in vitro. </i> ESCs and BCCs display signaling pathway convergence, which is highly and precisely regulated in ESCs and dysregulated in BCCs. Gene expression at the mRNA and protein level within restricted human BCCs indicates inhibition of the oncogenic EGFR and canonical Wnt/&beta;-catenin signaling pathways. Naked cuticle 2 (NKD2) is a potential point of cross-talk between these two pathways and its increased expression suggests a role in restored regulation of these pathways. Application of this model for mechanistic studies will enable development of a targeted treatment for triple negative human breast cancer.</p>

<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.