Master of Science / Department of Statistics / Gary L. Gadbury / Discerning the metabolic or enzymatic role of a particular gene product, in the absence of information indicating sequence homology to known gene products, is a difficult task. One approach is to compare the levels of metabolites in a wild-type organism to those in an organism with a mutation that causes loss of function of the gene. The goal of this project was to develop an approach to analyze metabolite data on wild-type and mutant organisms for the purpose of identifying the function of a mutated gene. To develop and test statistical approaches to analysis of metabolite data for identification of gene function, levels of 141 lipid metabolites were measured in leaves of wild-type Arabidopsis thaliana plants and in leaves of Arabidopsis thaliana plants with known mutations in genes involved in lipid metabolism. The mutations were primarily in fatty acid desaturases, which are enzymes that catalyze reactions in which double bonds are added to fatty acids. When these enzymes are mutated, leaf lipid composition is altered, and the altered levels of specific lipid metabolites can be detected by a mass spectrometry. A randomization P-Value and other metrics were calculated for all potential reactant product pairs, which included all lipid metabolite pairs. An algorithm was developed to combine these data and rank the results for each pair as to likelihood of being the actual reactant-product pair. This method was designed and tested on data collected on mutants in genes with known functions, fad2 (Okuley et al., 1994), fad3 (Arondel et al., 1992), fad4, fad5 (Mekhedov et al., 2000), fad6 (Falcone et al., 1994), and fad7 (Iba et al., 1993 and Gibson et al., 1994). Application of the method to three additional genes produced by random mutagenesis, sfd1, sfd2, and sfd3, indicated that the significant pairs for fad6 and sfd3 were similar. Consistent with this, genetic evidence has indicated that sfd3 is a mutation in the FAD6 gene. The methods provide a list of putative reactions for an enzyme encoded by an unknown mutant gene. The output lists for unknown genes and known genes can be compared to provide evidence for similar biochemical activities. However, the strength of the current method is that the list of candidate chemical reactions for an enzyme encoded by a mutant gene can be produced without data other than the metabolite profile of the wild-type and mutant organisms, i.e., known gene analysis is not a requirement to obtain the candidate reaction list.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Connexin proteins are the principle structural components of gap junction channels that connect the cytoplasm of two cells and maintain direct intercellular communication through the exchange of ions, small molecules and cellular metabolites. Colocalization and tissue-specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here the functions of a gap junction protein, connexin46 (Cx46), have been investigated in two hypoxic tissues, lens and breast tumor. We show that human breast cancer cells, MCF-7 and breast tumor tissues express connexin46 (Cx46) and it plays a critical role in protecting cells against hypoxia-induced death. Interestingly, I find that Cx46 is upregulated in MCF-7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes cell death of human lens epithelial cells (HLEC) and MCF-7 cells under hypoxic conditions. Furthermore, direct injection of anti-Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. Our result suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy against hypoxia. In the last part of the dissertation, we show that over expression of Cx46 induces the degradation of another connexin, connexin43, in rabbit lens epithelial NN1003A cells. Over expression of Cx46 increases ubiquitination of Cx43. Moreover, the Cx46-induced Cx43 degradation is counteracted by inhibitors of proteasome. Taken together, these data indicate that the degradation of Cx43, upon Cx46 over expression, is mediated by the ubiquitin-proteasome pathway. I also provide evidence that that C-terminal tail of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx46 has a novel function in the regulation of Cx43 turnover in addition to its conventional role as a gap junction protein. This may contribute to protection from hypoxia in both the lens and tumors.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / ADAMs (a disintegrin and metalloprotease) can influence multiple cellular processes involved in normal development and pathogenesis. ADAM12 expression levels are elevated in many pathological conditions including cancer, cardiovascular disease, and muscle regeneration. Recently, ADAM12 has emerged as a candidate cancer gene in a comprehensive genetic analysis of human breast cancers. The regulation of ADAM12 expression is poorly understood. Identification of new substrates for ADAM12 metalloprotease can expand our knowledge of processes in which ADAM12 is involved. Here, we show that ADAM12 expression is upregulated by transforming growth factor beta (TGF-beta), an essential signaling pathway for many cellular processes. This upregulation requires proteosomal degradation of a transcriptional repressor SnoN. Furthermore, breast cancer cell lines expressing high levels of SnoN have significantly impaired induction of ADAM12 by TGF-beta, suggesting an inverse correlation between SnoN and the extent of regulation of ADAM12 by TGF-beta. Additionally, we demonstrate that ADAM12 is one of the metalloproteases involved in shedding a Notch ligand, Delta like 1 (Dll1). The Notch signaling pathway plays a crucial role in cell fate decision during development and in adults. Cleavage of Dll1 by ADAMs occurs in cis and results in activation of Notch signaling in a cell-autonomous manner. Furthermore, the intracellular domain of Dll1 created after cleavage further enhances TGF-beta signaling in response to TGF-beta. Our analysis of breast cancer-associated mutations in the ADAM12 gene showed a lack of proper proteolytic processing of the ADAM12 protein and its mislocalization to the endoplasmic reticulum. Additionally, ADAM12 mutants show a dominant-negative effect on the processing of the wild-type ADAM12 and result in loss of the functional ADAM12 at the cell surface. Collectively, our results indicate a new mechanism of regulation of ADAM12 expression, expand the role of ADAM12 in the regulation of Notch signaling, and characterize cancer-associated mutations in the ADAM12 gene.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The common choice of medium for culturing pig umbilical cord matrix stem cells (PUCs) is high glucose Dulbecco’s Minimum Essential Medium (HG-DMEM) supplemented with fetal bovine serum (FBS). FBS is a chemically undefined supplement that encourages attachment of explants and cells and is useful for long-term proliferation in an undifferentiated state. Removing FBS from the culture medium would decrease the possibility of microbial contamination and might produce more consistent results. A defined medium would facilitate experiments to determine requirements for specific growth factors and nutrients. Starting PUCs in a FBS-free environment proved to be a challenge. The results of 15 experiments testing various media, supplements, and culture conditions indicate that PUCs initially plated in an FBS-free environment do not attach as readily as those in HG-DMEM supplemented with FBS. PUCs were collected using enzyme digestion of the whole cord or by plating explants from the cord in culture medium. In the final experiment PUCs were seeded in 24-well plates (5.0 * 10[superscript]4 viable cells per well) with a collagen coating and cultured in Knock-out DMEM (KO-DMEM) with basic fibroblast growth factor (5ng/mL) and platelet derived growth factor (5ng/mL) in a low oxygen atmosphere (5% O[subscript]2/ 5% CO[subscript]2/ 90% N[subscript]2). The total non-adherent cell count at passage 1 was 1.78 * 10[superscript]5 +or- 3.68 * 10[superscript]4 and the total adherent cells were 2.58 * 10[superscript]5 +or- 9.29 * 10[superscript]4. The well confluence during initial cell proliferation appeared similar to cells cultured in the control media with 20% FBS (total adherent cells = 6.40 * 10[superscript]5 +or- S.E. 1.61 * 10[superscript]5 and total non-adherent cells = 2.88 * 10[superscript]5 + 7.60 * 10[superscript]4). However the number of adherent cells recovered for passage 2 was considerably less for cultures in FBS-free media than for the control group. Serum may affect attachment by providing attachment factors or it could change expression of integrins or other attachment molecules on the PUCs that enhance attachment to plastic or other substrates. In future studies the requirements for attachment of PUCs should be further evaluated.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / The avascular lens tissue depends on the gap junction channels to facilitate intercellular communication for supplying cells deep within the lens with nutrients and removing waste products of cellular metabolism. In the absence of the protein synthesis machinery in the inner lens fiber cells, the proper regulation of gap junction channels becomes extremely important as disturbance of the lens homeostasis can lead to cataract development. Phosphorylation of gap junction subunit connexin proteins has been shown to play an important channel-modulating role in a variety of tissue. Protein kinase C-[Gamma] (PKC[Gamma]) has been implicated in the phosphorylation of connexins in the lens. Here the role of PKC[Gamma] in the regulation of gap junction coupling in the mouse lens has been investigated. We have compared the properties of coupling in lenses from wild type (WT) and PKC[Gamma] knockout (KO) mice. Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. PKC[Gamma]was found to regulate the amount and distribution of Cx43 in the lens. Gap junction coupling conductance in the differentiating fibers (DF) of PKC[Gamma] KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. Absence of PKC[Gamma] in the KO mice also caused abnormal persistence of nuclei in the typical nucleus-free region in the DF. These results suggest a major role for PKC[Gamma] in the regulation of gap junction expression and coupling in the normal lens mediated by phosphorylation of the lens connexins. This becomes very vital in the diabetic lenses which contain a depleted amount of PKC[Gamma] and people suffering from spinocerebellar ataxia type-14 (SCA14) who have a mutated inactive form of PKC[Gamma]. Prolonged exposure of lenses to oxidative stress in these patients can lead to cataract formation. In cultured human lens epithelial cells (HLECs), 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the depletion of Cx43 protein level via PKC-mediated phosphorylation of Cx43. At the same time Cx46 protein and message levels were upregulated in response to TPA treatment. So, the PKC activator regulates Cx43 and Cx46 in opposing ways. The possible mitochondria localization of Cx46 reported here could help in finding the non-junctional roles for Cx46.

Master of Science / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / Peptides based on the second transmembrane segment of the glycine receptor (M2GlyR) were made to provide a potential therapeutic treatment for cystic fibrosis (CF) and a latent absorption enhancer for drug delivery. For similarity of presentation, unique synthetic peptide sequences have been given alpha-numeric designations. Results are presented from studies focusing on four peptides. In the first study, the contributions of synthetic peptides p1171, p1172 and p1173 to net transepithelial ion transport were measured as a first step toward the goal of testing whether pore length or electrostatics of pore lining residues will affect anion transport. Peptide p1130 exhibits many attributes that make it an ideal synthetic peptide for CF treatment, but has low permselectivity for anions. Therefore, it is used as a platform for modification. Peptide p1171 is doubly substituted with diaminopropionic acid at positions T13 and T17. Peptide p1172 and p1173 are separately one and two helical turn(s) inserted into the p1130 backbone. Apical exposure of MDCK monolayers to these peptides caused a rapid increase in short circuit current (Isc), an indicator of net ion transport. The increase in Isc caused by p1172 or p1173 was accompanied by increase in transepithelial electrical conductance (gte). The electrophysiological results suggested that these modified peptides can assemble in the apical membrane of epithelial cells to form functional ion-conducting pores. Peptide NC-1059, which provides for ion transport across epithelial cells derived from many sources, was studied further to assess cellular changes that account for increased gte. NC-1059 increased Isc, gte and enhanced permeation of dextrans in a concentration dependent manner. Results from previous and current studies show that NC-1059 modulated the epithelial paracellular pathway by altering the distribution and abundance of junctional proteins. Immunoblotting and immunolabeling with confocal microscopy showed that NC-1059 induces reorganization of actin and causes a reduction in F-actin abundance in epithelial cells. The distributions were changed and cellular abundances were reduced of tight junction proteins occludin and ZO-1 and adherens junction proteins E-cadherin and β-catenin by NC-1059. These effects were largely reversed in 24 hr and fully recovered in 48 hr. Therefore, NC-1059 has the therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

Doctor of Philosophy / Department of Biology / Stephen K. Chapes / Silvia Mora Fayos / My work focused on understanding adipocyte function and regulation because of the importance to diabetes. In addition to being a fat storage depot, adipose tissue is an endocrine tissue. Adiponectin and leptin are two adipokines that control insulin sensitivity and energy balance. In spite of their importance, there are still questions about their secretion. I hypothesized that leptin and adiponectin follow different secretory routes. I found adiponectin localized in Golgi and the trans Golgi Network, while leptin mostly localized in ER during basal metabolisms. Common requirements for their secretion were the presence of class III Arf proteins and an intact Golgi apparatus, since BFA treatment inhibited secretion of both adiponectin and leptin. I found that trafficking of adiponectin is dependent on GGA1 coated vesicles. Endosomal inactivation significantly reduced adiponectin, but not leptin, secretion in both 3T3L1 and isolated rat adipocytes. Also, adiponectin, but not leptin, secretion was reduced in cells expressing non- functional form of Rab11 and Rab5 proteins. However, secretion of leptin, but not adiponectin was inhibited in cells expressing mutants of Protein Kinase D1. These results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and pathways. Insulin resistance is associated with macrophage infiltration into adipose tissue and elevated levels of IL-6, TNF-alpha and IL-1beta Therefore, the second part of my dissertation tested the hypothesis that the interaction of macrophages and adipocytes causes insulin resistance. To test this hypothesis, I co-cultured macrophages and adipocytes. I found that mouse elicited peritoneal macrophages significantly decreased insulin-stimulated GLUT4 translocation to the plasma membrane in a contact-independent manner. IL-6 was the most inhibitory cytokine in reducing GLUT4 translocation, GLUT4 expression, Akt phsphorylation and reducing adipocyte differentiation compared to TNF-alpha and IL-1beta. These data suggest that IL-6 is the most effective cytokine secreted by macrophages involved in insulin resistance. Lastly, I tested the impact of adipocytes on macrophage differentiation in vitro and in vivo. I found that C2D macrophages isolated from the peritoneal cavity had increased IL-6 transcript levels after co-culture with 3T3L1 adipocytes in vitro. After i.p. injection, C2D macrophages isolated from WAT increased expression of mature macrophage surface markers and transcript levels of proinflammatory cytokines compared to C2D cells in vitro. However, macrophages isolated from BAT expressed low levels of cytokines and macrophage surface markers.

Doctor of Philosophy / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells; Broughman, J. R. et al; Am J Physiol Cell Physiol 286: C1312-23). In this first study experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Human (T84, Calu-3) and non-human (IPEC-J2, PVD9902) epithelial cells derived from intestinal (T84, IPEC-J2), airway (Calu-3), and genitourinary (PVD9902) tissues were grown on permeable supports. Ion transport and barrier function were assessed electrically in a modified Ussing chamber. Basal short circuit current (I[subscript sc]) was typically less than 3 [Mu]A cm[superscript-2]. Apical NC-1059 exposure caused, in all cell types, an increase in I[subscript sc] to >15 [Mu]A cm[superscript-2], indicative of net anion secretion or cation absorption that was followed by an increase in transepithelial conductance (g[subscript te] in mS cm[superscript-2]; T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.2 to 13). NC-1059 induces a concentration dependent change in the I[subscript sc] and g[subscript te] across these epithelia. The results in all cases were consistent with both a transcellular and a paracellular effect of the peptide. NC-1059 enhanced permeation of dextrans ranging from 10 kDa to 70 kDa across all epithelia tested. These results document an effect of NC-1059 on the paracellular route of epithelial barriers. Immunolabeling, confocal microscopy and immunoblotting methods were used in a second study to assess the molecular changes associated with increased paracellular permeability. NC-1059 induced a substantial reorganization of actin within 60 minutes of exposure. Confocal microscopy revealed that the changes in actin organization were accompanied by a pronounced change in the abundance and distribution of tight junction proteins occludin and ZO-1. Immunoblotting results suggest a time and concentration dependent effect on cellular abundance of these tight junction proteins. The effects on g[subscript te] and junctional proteins are transient with > 85% of recovery in 24 hours post exposure and full recovery within 48 hours. The reversible modulation of the epithelial tight junctions has therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / Muscle satellite cells are the primary stem cells of postnatal skeletal muscle. Quiescent satellite cells become activated and proliferate during muscle regeneration after injury. They have the ability to adopt two divergent fates: differentiation or self-renewal. The Notch pathway is a critical regulator of satellite cell activation and differentiation. Notch signaling is activated upon the interaction of a Notch ligand present in a signal-sending cell with a Notch receptor present in a signal-receiving cell. Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. In this study, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewing satellite cells. We also demonstrated that Dll1 represents a substrate for several ADAM metalloproteases. Dll1 shedding takes place in a pool of Pax7-positive self-renewing cells, but Dll1 remains intact in differentiated myotubes. Inhibition of Dll1 shedding with a dominant-negative form of ADAM12 leads to elevated Notch signaling, inhibition of differentiation and expansion of the pool of self-renewing cells. We propose that ADAM-mediated shedding of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. Pax7 plays a key role in protecting satellite cells from apoptosis. The mechanism of Pax7 protecting muscle satellite cells from apoptosis is not well understood. In the second part of this study, we show that Pax7 up-regulates manganese superoxide dismutase (MnSOD) at the transcriptional level, suggesting the involvement of MnSOD in Pax7-mediated cell survival. A specific chromosomal translocation involving the Pax7 gene and generation of a fusion protein Pax7-FKHR is found a childhood cancer, rhabdomyosarcoma. Furthermore, the level of the wild-type Pax7 is down-regulated in rhabdomyosarcomas. In the third part of this dissertation, we investigated the dominant-negative effect of Pax7-FKHR fusion protein on the wild-type Pax7, and found that the Pax7 protein level is down-regulated by Pax7-FKHR expression while the Pax7 mRNA level is not affected. We propose a specific microRNA-mediated inhibition of Pax7 mRNA translation by the oncogenic Pax7-FKHR fusion protein.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Apoptosis is an extremely conserved process among metazoans. This dissertation will describe apoptotic regulation in two orders of insects, Lepidoptera and Diptera. In the lepidopteran host Trichoplusia ni, we describe phenotypes of infection with the baculovirus AcMNPV lacking the caspase inhibitor gene P35. In the lepidopteran host Spodoptera frugiperda, infection with this mutant virus results in apoptosis, which dramatically hinders spread of the virus in the host. In T. ni, however, infection with this mutant virus is similar to wild-type with normal spread, but the end result of liquefaction does not occur. Experiments indicated that infection of T. ni cells with the P35 mutant virus (P35Δ) resulted in caspase activation, and the P35Δ virus lacked the ability to inhibit these active caspases. With the P35Δ virus a slower entry phenotype was observed, but when the P35Δ virus was grown in the presence of a caspase inhibitor the entry phenotype was rescued. This indicated that caspases have detrimental effects on budded virions, and illustrated that P35 is needed to make robust virions. With regards to Diptera, apoptosis-regulatory genes were annotated in the yellow fever mosquito, Aedes aegypti. The genes annotated included multiple caspases and caspase regulators. Phylogenetic relationships were determined among the caspases from Anopheles gambiae, Ae. aegypti and Drosophila melanogaster, expression patterns were determined for all the annotated genes in Ae. aegypti, and one of the genes, an IAP antagonist named IMP, was functionally characterized. Further characterization of the phylogenetic relationships of caspases from fifteen dipteran species was performed by obtaining gene models for caspases of recently sequenced genomes for twelve Drosophila species and three mosquito species. Furthermore, several Drosophila and mosquito species were found to contain caspase genes with substitutions in critical active site residues. These genes were proposed to encode caspase-like decoy molecules. While these have been found in humans and nematodes, this is the first report for these molecules in insects. One of the caspase-like decoy molecules was found to increase the activity of its paralog caspase.