Browsing by Author "Barbara Sherry, Committee Member"

Filter results by typing the first few letters
Now showing 1 - 6 of 6
  • No Thumbnail Available
    C/EBP-alpha is an epithelial tumor suppressor gene, and mitogenic stimulation reciprocally regulates C/EBP-alpha and C/EBP-beta
    (2008-11-09) Loomis, Kari Danielle; Robert C. Smart, Committee Chair; Barbara Sherry, Committee Member; David Bird, Committee Member; Marcelo L. Rodriguez-Puebla, Committee Member
    CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ are basic leucine zipper transcription factors that function in the inhibition and stimulation of cell cycle progression, and regulate differentiation in various cell types. C/EBPα is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. While C/EBPα mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPα occurs in numerous human epithelial cancers including those of lung, liver, endometrium, skin and breast suggesting a possible tumor suppressor function. However, direct evidence for C/EBPα as an epithelial tumor suppressor is lacking due to the absence of C/EBPα mutations in epithelial tumors and the lethal effect of C/EBPα deletion in mouse model systems. To examine the function of C/EBPα in epithelial tumor development, an epidermal-specific C/EBPα knockout mouse was generated. The epidermal-specific C/EBPα knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation or apoptosis; demonstrating that C/EBPα is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPα knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate and the rate of malignant progression. Mice hemizygous for C/EBPα displayed an intermediate enhanced tumor phenotype. Our results suggest decreased expression of C/EBPα contributes to deregulation of tumor cell proliferation. C/EBPα had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPα blocked Ras-induced and epidermal growth factor–induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Next the effects of mitogenic stimulation on C/EBPα and C/EBPβ expression were studied. Stimulation of cells in culture with EGF resulted in decreased C/EBPα expression, as well as a reciprocal increase in C/EBPβ expression. Further, loss of C/EBPβ results in an inhibited transition from the G1 to S phase of the cell cycle after treatment with EGF, and this reduction was associated with decreased expression of E2F target genes. Both C/EBPα and C/EBPβ have been proposed to affect cell cycle progression through interactions with E2F. We observed that C/EBPα blocked while C/EBPβ stimulated Ras- and EGF-induced E2F activity in keratinocytes. My results demonstrate that C/EBPα is dispensable for epidermal homeostasis, that C/EBPα and C/EBPβ are reciprocally regulated by mitogenic stimulation, and provide genetic evidence that C/EBPα is a haploinsufficent suppressor gene in epithelial tumorigenesis.
  • No Thumbnail Available
    Cis-acting Elements Important for Potato Virus X Minus-strand RNA Synthesis In Vitro and In Vivo
    (2007-02-27) Hu, Bin; Paul Wollenzien, Committee Member; Cynthia L. Hemenway, Committee Chair; Barbara Sherry, Committee Member; E. Stuart Maxwell, Committee Member
    In order to identify cis-acting elements required for Potato virus X (PVX) minus-strand RNA synthesis, replication in in vitro and in vivo systems was compared. Specifically, RNA dependent RNA polymerase activity using a 850 nt template in PVX infected tobacco plant extract and using infectious full-length transcripts in tobacco protoplasts was analyzed. To facilitate quantitation of results from the in vitro system, the RdRp assay was optimized in several ways. Initial experiments showed that processing of extracts from fresh plant tissue was optimal for the in vitro RdRp assay. Purification of nuclease Bal31 with stable RNase activity was critical for consistency in making and assaying template-dependent plant extracts. Optimal salt concentrations, reaction volume, incubation time, and template concentration conditions were found to ensure template specificity and quantifiable product levels for PVX RdRp. Comparison of data obtained with this optimal extract and the protoplast system showed that the conserved hexanucleotide element and conformation of stem-loop 3 are required for minus-strand RNA synthesis both in vitro and in vivo. More strikingly, we found that long-distance RNA-RNA interactions between conserved internal elements and the hexanucleotide element are required for optimal minus-strand RNA synthesis both in vitro and in vivo. In addition, multiple internal elements can serve as interaction partners. Thus, similar to plus-strand RNA synthesis, PVX minus-strand RNA synthesis requires local elements and long-distance RNA-RNA interactions. A model for RNA synthesis is proposed in which both termini of the genome are paired with internal elements.
  • No Thumbnail Available
    Mining of cis-Regulatory Motifs Associated with Tissue-Specific Alternative Splicing
    (2009-08-11) Kim, Jihye; Steffen Heber , Committee Chair; Zhao-Bang Zeng, Committee Member; Barbara Sherry, Committee Member; Eric A. Stone, Committee Member
    Alternative splicing (AS) is an important post-transcriptional mechanism that increases protein diversity and may affect mRNA stability and translaftion efficiency. Despite its importance, our knowledge about its mechanism and regulation is very limited. Although it is known that the regulation of AS is influenced by multiple factors, most previous studies have focused on analyzing an individual regulator. In this dissertation, we apply three types of association rule mining techniques to discover cis-regulatory motifs or motif groups that are associated with specific AS patterns in mouse. General association rule mining for categorical attributes is used to find “motif=>motif†rules in gene groups that show similar exon skipping patterns. This method provides candidates for interacting motifs. Discretization-based and distribution-based quantitative association rule mining techniques are used to find “motif => exon skipping profile†rules. Many of the discovered motif candidates coincide with known splicing factor binding sites. Our ultimate goal is to find motifs and motif combinations that are involved in the dynamic regulation of AS. Based on our observations we hypothesize that some cis-regulatory elements affect AS only in combination with other elements. Interacting motifs show interesting differences to motifs that act individually. For example, interacting motif pairs are more conserved, they occur on average closer to the splice sites, motif pairs derived from distribution-based association rule mining, occur also in higher multiplicity. Based on these observations, we hypothesize that interacting cis-regulatory motifs might often correspond to weaker binding sites that occur in clusters close to the regulated splice sites.
  • No Thumbnail Available
    Protein Evolution From Sequence To Structure.
    (2003-09-01) Buck, Michael Joseph; Jeff Thorne, Committee Member; Barbara Sherry, Committee Member; Michael Purugganan, Committee Member; William R. Atchley, Committee Chair
    The purpose of this research is to elucidate how natural selection shapes protein evolution. The question was addressed by exploring protein sequence evolution, 3D structural evolution, and analysis of the multidimensional nature of amino acid covariation. This thesis begins with a study of protein sequence evolution. 118 different bHLH genes in the completely sequenced Arabidopsis thaliana genome and 131 bHLH genes in the rice genome were identified and characterized using phylogenetic analysis. These plant proteins were classified into 15 distinct plant clades and were under weaker selective constraints than their animal counterparts. Additionally, it was shown that lineage specific expansions and subfunctionalization have fashioned regulatory proteins for plant specific functions. To further characterize the bHLH domain, a canonical 3D structure was created from solved structures. This canonical structure was used as a template for producing 3D models for other representative bHLH proteins, which were then compared, contrasted, and grouped based on structural characteristics. Structural similarities were discovered within the bHLH domain between three clades (Max, Myc, and PbHLH-LZ). In addition, structural models of the Sat proteins suggest a strong similarity to other bHLH proteins, which is in disagreement with previous functional characterization. To further understand the dimensionality of protein evolution, the independence of amino acid sites was explored using multivariate factor analysis. A matrix of pairwise normalized mutual information values were computed among amino acid sites for the serpin proteins. The normalized mutual information matrix was partitioned into orthogonal dimensions by factor analysis. Each eigenvector from the factor analysis can be interpreted as having phylogenetic or structural/functional explanations or combinations of both. This approach discerns strong amino acid covariation within several key functional regions including the RCL, shutter, and breach. In addition, this approach elucidates hydrogen bonding, hydrophobic, and electrostatic interactions within the serpin protein family.
  • No Thumbnail Available
    Role of the Cytoplasmic Tail of Equine Infectious Anemia Virus Transmembrane Glycoprotein in Acute Disease Induction
    (2004-12-26) Jia, Bin; Barbara Sherry, Committee Member; Scott M. Laster, Committee Member; Wayne A. Tompkins, Committee Member; Frederick J. Fuller, Committee Chair
    Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus of horses. EIAV is unique among lentiviruses in that a further cleavage event occurs within the N-terminus of the cytoplasmic tail (CT) of transmembrane (TM) glycoprotein and yields a C-terminal non-glycosylated p20 protein. The p20 comprises more than two-third of the CT domain and contains both of the amphipathic α-helices. To test the role of the EIAV CT domain in acute disease induction, we constructed a p20-truncated clone (p19/wenv17Δ20) on the background of a highly virulent EIAV infectious clone p19/wenv17 by introducing three termination codons into the N-terminal coding region of p20. The derived virus replicated at a delayed and lower level compared with that of parental virus in equine macrophages in vitro. In vivo, the p19/wenv17Δ20 virus showed attenuation and did not induce acute disease like the parental (p19/wenv17) virus. The viral load in ponies infected by p19/wenv17Δ20 virus was about 10-1000 fold lower than that of ponies infected by parental (p19/wenv17) virus. In vitro studies on the properties of the p20-truncated virus showed that truncation of the p20 did not impair the envelope glycoprotein incorporation into virions. There was also no severe defect in virus replication. The delayed and lower level replication of p20-truncated virus compared with parental virus was most probably due to small delays in several steps in the virus life cycle. In addition, p20 expressed in trans could not compensate for the absence of p20 in the p20-truncated virus.
  • No Thumbnail Available
    West Nile Virus Nonstructural Protein 1 Modulation of Innate Immunity.
    (2011-03-22) Wilson, Jason R.; Frank Scholle, Committee Chair; Barbara Sherry, Committee Member; Ian Petty, Committee Member; DE SILVA, ARAVINDA (UNC-CH), Committee Member; Scott Laster, Committee Member