Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviridae is the only known virus family that encodes genes with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting cell growth, differentiation, and motility. The role of viral fgfs during infection is not known. In this study, we investigated gene regulation and function of the Autographa californica M nucleopolyhedrovirus (AcMNPV) fgf during infection of permissive insect cells. We demonstrated that the AcMNPV fgf, vfgf, was transcribed as a 0.6-kb mRNA at early times post infection, but as part of a 1.4-kb bicistronic mRNA at late times. To determine its function, we examined common characteristics between vFGF and other well-characterized FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate chemokinesis of different types of insect cells. We also constructed a recombinant of AcMNPV lacking a functional vfgf and analyzed it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in two cell lines and at both high and low multiplicities of infection. In addition, we observed no obvious differences in the viral DNA synthesis and the protein kinetic profiles of cells infected with the mutant and parental viruses. Finally, coinfection of vfgf-containing and -deficient viruses and their passage for several generations did not reveal a consistent growth advantage for either virus. We propose that vFGF is the signal that directs the motility of uninfected tracheal or blood cells to infected tissues, enabling the virus to infect additional cells and spread systemically in the insect host. This proposal may explain a dispensable role for vfgf during virus infection in cell culture; nonetheless, we expect a distinct phenotypic difference between vfgf-deficient and vfgf-containing viruses during infection in the insect host.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Members of the protein super family of tetraspanins are best defined by a simple structure comprising four transmembrane domains, two extracellular loops of unequal size, and short cytoplasmic regions. Despite their small size, tetraspanins are able to participate in multiple functions, as diverse as B cell activation, cancer metastasis, and viral infection. To compensate for a lack of intrinsic enzymatic activity, tetraspanins have gained the fascinating ability of associating with numerous different proteins. In addition, tetraspanins interact with each other forming a network on the plasma membrane: the tetraspanin web. In this way, functionally related proteins binding to different tetraspanins can be brought into close vicinity, thereby enhancing signaling pathways. The tetraspanin web is a dynamic environment and its regulation has grasped the attention of several research groups in the past few years. Particularly, several tetraspanins have been found to be palmitoylated, a post-translational modification attaching a palmitic acid to cysteine residues in a reversible manner. Palmitoylation is thought to be important for the integrity of the tetraspanin web. We examined the effect of disrupting putative palmitoylation sites on the tetraspanin CD81 by mutating its juxtamembrane cysteines. By flow cytometry, we observed a decrease in the detection of mutant CD81 at the cell surface. This was not due to defects in protein trafficking or antibody affinity, and might reflect an abnormal CD81 distribution in a membrane environment that prevents the exposure of the epitope recognized by the CD81 antibody. Immunoblotting analysis revealed a novel CD81 processing event that was impaired in the mutant CD81 proteins compared to wild-type. Finally, co-immunoprecipitation assays showed a reduction in binding of tetraspanin CD9 and Ig superfamily member EWI-2 to mutant CD81. Taken together, these results suggest the importance of juxtamembrane cysteines (via palmitoylation or protein conformational changes) in protein interactions of CD81 within the tetraspanin web. Although 33 tetraspanins are expressed in humans, less than half of them have been well studied. Among the “orphan” tetraspanins awaiting further examination is Tspan-2. Here, we provide the first elements for the characterization of mammalian Tspan-2 by investigating expression patterns, N-glycosylation status, and association with other tetraspanins.

Master of Science / Department of Biochemistry / Dolores J. Takemoto / The vertebrate lens relies on gap junction-mediated intercellular communication to maintain cellular homeostasis and lenticular transparency. Differentiation of cuboidal lens epithelial cells into cortical fiber cells involves the degradation of endogenous gap junction protein, connexin 43 (Cx43) and the up-regulation of connexin 46 (Cx46). Cx46 may also be involved in the hypoxia response in other tissues; a function that can possibly be attributed to unique phosphorylation sites at the cytoplasmic C-terminus. In this study, we have developed a mammalian (rabbit) lens epithelial cell (RLEC) culture model that overexpresses Cx46 to ascertain the role of Cx46 in differentiation and oxidative stress response. The cell line N/N1003A was stably transfected with a GFPCx46 plasmid construct, and analyzed for differentiation markers including endogenous gap junction protein isoforms (Cx43 and Cx50). Western blot analysis and visual observation determined that the stable overexpression of Cx46 (sCx46OE) induced the degradation of Cx43 and elicited morphological changes indicative of fiber cell elongation. Total RNA from RLEC culture was isolated and analyzed for mRNA levels using RT-PCR. Comparable levels of Cx43 transcript were present in wild type, transient Cx46OE (tCx46OE), and sCx46OE which suggests a post-transcriptional regulation of Cx43 degradation. Treatment of sCx46OE with proteasome inhibitors restored Cx43 protein levels, and scanning confocal microscopy supported our hypothesis that Cx43 is degraded in differentiating lens cells by way of a ubiquitinmediated proteasomal pathway. It is our conclusion that Cx46 has application in hypoxic conditioning and differentiation in addition to its conventional role as a gap junction protein.

Master of Science / Department of Clinical Sciences / James W. Carpenter / Current in vitro maturation (IVM) systems for domestic dog oocytes are inefficient, largely due to the species' unique reproductive physiology. The size of donor follicle influences developmental competence of dog ovarian oocytes. Specifically, oocytes from follicles > 2 mm in diameter complete in vitro nuclear maturation at a higher rate than those from smaller follicles. The objective was to determine the influences of follicular size, maturation time, and meiotic status on oocyte metabolism. We hypothesized that metabolic patterns differed between oocytes from small versus large follicles. Oocytes (n = 531) from adult ovaries were collected and grouped based on follicular size (small, < 1 mm, n = 252; medium, 1 to 2 mm, n = 231; and large, > 2 mm, n = 48). Oocytes were cultured for 0, 24, or 48 hours at 38.5°C in 5% CO[subscript]2 in 80 [Mu]L of TCM 199 + 25[Mu]M [Beta]- mercaptoethanol + 10 ng/ml epidermal growth factor + 0.25 mM pyruvate + 2.0 mM glutamine + 0.1% polyvinyl alcohol + 0.03 mg/ml streptomycin + 0.03 mg/ml penicillin G sodium (IVM medium), assessed for metabolism and evaluated for nuclear status. For metabolic assessments, oocytes were incubated for 3 h in 3 [Mu]l of IVM medium containing (1) 0.005 mM [0.064 [Mu]Ci/[Mu]l] D-53H-glucose (glycolysis) + 1 mM D-614C [0.053 [Mu]Ci/[Mu]l] glucose (glucose oxidation) or (2) 0.001 mM [0.041 [Mu]Ci/[Mu]l] L-G-3H-glutamine + 1 mM [0.027 [Mu]Ci/[Mu}l] 1-14C pyruvate, placed on the lid of a centrifuge tube containing 25 mM NaHCO3 and trapped radioactivity was measured using a [Beta]-counter. Only oocytes at an appropriate meiotic stage for each culture period (n = 380) were included in data analysis (e.g., germinal vesicle stage at 24 and 48 h culture were excluded). Differences in metabolism among groups were analyzed by ANOVA (main effects being follicular class, culture interval, and meiotic status). Oocytes recovered from large follicles metabolized significantly more pyruvate, glutamine, and glucose (via glycolysis) than those from small ones (p < 0.05). Across meiotic stages and follicular sizes, glycolytic rate was lowest in oocytes cultured for 24 hours (p < 0.05) compared to 0 or 48 hours. Metaphase II oocytes had a significantly higher glycolytic rate than those at other meiotic stages (p < 0.05). At culture onset (0 h), oocytes from small follicles predominately used pyruvate (p < 0.05), while oocytes from larger follicles (p < 0.05) predominately metabolized glucose. The present data suggests that dog oocytes preferentially use glucose as an energy substrate and that increasing glycolytic rate correlates with meiotic maturation. In addition, oocytes collected from large follicles exhibit increased metabolic capabilities that may be responsible for their increased developmental competence during IVM.

Master of Science / Department of Grain Science and Industry / Praveen V. Vadlani / Synthesis of chemical derivatives from finite fossil fuels requires considerable energy inputs and leaves an undesirable environmental footprint. The emerging biorefinery approach leads to sustainable processing of biomass into a wide spectrum of bio-based products, catering to food, feed, chemicals, materials, and bioenergy industries. Cyanophycin (multi-L-arginyl-poly-L-aspartic acid, CGP) is a non-ribosomally synthesized reserve polypeptide, which consists of equimolar amounts of arginine and aspartic acid arranged as a polyaspartate backbone and arginine as the side chain. Cyanophycin is a source of the constituent N-functionalized platform chemical, which can be further processed into many other chemicals of importance. It can be hydrolyzed in mild condition to its constituent amino acids - aspartic acid and arginine. These amino acids may be utilized directly in food and pharmaceutical applications. Based on the chemical structure of these amino acids and the presence of functionalized nitrogen-containing groups, it is conceivable that a number of industrial chemicals can be synthesized, for example: 1, 4-butanediamine， a co-monomer in the production of nylon-4, 6. Other chemicals which could be obtained from cyanophcyin, that are currently prepared from fossil resources, include 1,4-butanediol and urea. Cyanophycin can also be hydrolyzed to a derivative with reduced arginine content or even to poly-aspartic acid, and used as a biodegradable substitute for synthetic polyacrylate in various technical process, such as water treatment (water softeners) and plastics. Cyanophycin is produced by most cyanobacteria in nature; however, these microbes are not suitable for large-scale production due to slow growth and low polymer content. Biosynthesis of cyanophycin is catalyzed by a single enzyme - cyanophycin synthetase (CphA), which is encoded by cyanophycin synthetase structure gene (cphA). The cphA gene can be expressed in several bacteria and plants. E.coli is one of the most commonly used bacterial hosts for the production of recombinant proteins. The recombinant culture has the ability to produce considerably large quantities of cyanophycin in a shorter period of time compared to cyanobacteria. Genome of Anabaena variabilis ATCC 29413 has been sequenced, and it contains the structural gene (cphA) for cyanophycin synthetase. The native enzyme-cyanophycin synthetase from this cyanbacterium culture had been purified and it consists of identical subunits of 98kDa. Polymerization of the amino acids to cyanophycin in vitro is dependent on the presence of ATP, K+, Mg2+, a (cyanophycin) primer and a thiol reagent such as β-mercaptoethanol in the reaction mixture. Our research is the first time that cphA gene from A. variabilis ATCC 29413 was cloned into E.coli. The 2.7 kb cphA gene was amplified by PCR, ligated to the vector pET45b+ and cloned into BL21 (DE3) pLysS and BL21 (DE3). Characterization of cyanophycin was performed by SDS-PAGE, HPLC, mass spectrometry and amino acid analysis. Results showed that protein with molecular weight of 21.5 to 31 kDa did not match any E.coli proteins when compared with E.coli protein data base, thereby showing expression of a protein foreign to host strain. Amino acids analyses showed that the cyanophycin produced by recombinant E.coli contained aspartic acid and arginine, and small amount of lysine, in the ratio: 1.05: 1: 0.2 (mass basis), thereby confirming cyanophycin biosynthesis. Experiments for high cyanophycin synthesis was performed at shake flask and 2-L fermentor level using recombinant BL21 (DE3) pLysS, LB broth as carbon and nutrient source, and casamino acids as primer. The maximum yield of cyanophycin obtained in flask level was 7.6% of cell dry mass, and the yield increased to 12.6% of cell dry mass at 2-L fermentor level. Cyanophycin is also referred to as “structural granules” because of substructures visible in electron micrographs. Phase contrast photomicrograph was able to depict cyanophycin inclusions in the cytoplasm, and transmission electron microscopy depicted finer details inside cell after IPTG induction.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Localized inoculation of a plant with an avirulent pathogen results in the activation of systemic acquired resistance (SAR), a defense mechanism that confers enhanced resistance against a variety of pathogens. The activation of SAR requires the translocation of an unknown signal from the pathogen-inoculated organ to the other organs where defenses are primed to respond faster in response to a future attack by a pathogen. Previous studies with the Arabidopsis thaliana dir1 (defective in induced resistance1) and sfd1 (suppressor of fatty acid desaturase deficiency1) mutants implicated a role for plant lipids in the activation of SAR. DIR1 encodes a putative lipid transfer protein and SFD1 encodes a dihydroxyacetone phosphate (DHAP) reductase involved in plastid glycerolipid metabolism. To further evaluate the role of DHAP reductases and plastid lipids in SAR, the involvement of two additional putative DHAP reductase encoding genes (AtGPDHp and AtGPDHc) and the SFD2 gene, which like SFD1 is involved in plastid glycerolipid metabolism, in SAR was evaluated. Only SFD2 was found to be essential for SAR. Although the lipid profile of the sfd2 mutant was similar to that of the fad5 (fatty acid desaturase 5) mutant, sfd2 is not allelic with fad5 and does not influence FAD5 expression. The SFD2 gene was mapped to an 85 kilo basepairs (kb) region on the third chromosome of Arabidopsis. The lipid composition defect of the sfd2 mutant was partially complemented by two independent recombinant bacterial artificial chromosomes (BACs) that contained genomic DNA spanning the wild type SFD2 locus. The role of plastid synthesized glycerolipids in the activation of SAR was further evaluated by characterizing SAR in additional Arabidopsis mutants that were deficient in plastid lipid metabolism. The requirement of MGD1 (MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE 1), DGD1 (DIGALACTOSYL-DIACYLGLYCEROL SYNTHASE 1) and FAD7 (FATTY ACID DESATURASE 7) genes in SAR, confirmed the essential role of plastid glycerolipids, presumably a galactolipid-dependent factor, in signaling associated with the SAR.

Doctor of Philosophy / Department of Anatomy and Physiology / Frank Blecha / L Tonatiuh Melgarejo / Background - Cardiovascular disease (CVD) impacts the lives of millions, and ranks as the number one killer world-wide. Despite significant research efforts, CVD remains a major burden on the national health care system, and novel therapeutic modalities to effectively and curatively fight many debilitating diseases of the heart and vasculature are urgently needed. The role of inflammation in the development of CVD has been increasingly in focus through the past decade. Elucidating upon the plethora of innate immune mechanisms likely involved in CVD therefore becomes of immediate interest. Host defense peptides (HDPs) are central elements of innate immunity, encompassing molecules (including the defensin peptides) with wide-reaching biological effects, including immunomodulation and antimicrobial activity. Hypothesis & Specific Aims - The study's main hypothesis relies upon the basic concept that the heart possesses a local innate defense system, which actively aids in fighting off a variety of "danger signals", and that a disarray in this defense contributes to development of CVD. The heart expresses beta-defensin peptides (BDs), and we theorized that these HDPs act as a local defense system within the myocardium - or in other words as "guardians of heart health". The specific aims of the experimental studies were to 1) Evaluate expression of cardiac BDs in response to inflammatory mediators, and 2) Assess the functional properties (including antimicrobial activity and immunomodulation) of synthetic BD peptides in vitro. Design & Methods - To test our hypothesis, we studied myocardial beta-defensin expression (rBDs) in a rat model, comparing levels among two experimental and one control group. Animals were exposed to lipopolysaccharide (LPS) or high-fat diet (HFD) intake - representative of exposure to either an infectious (LPS) or non-infectious (HFD) inflammatory mediator. Serum samples were collected for measurement of cytokines, inflammatory and cardiac biomarkers and lipid-profiling. Beta-defensin levels were assessed using customized Superarray assays and qRT-PCR, and all amplicon sizes on the PCR products were subsequently confirmed using agarose gel electrophoresis. Serum levels were assessed on commercial ELISA kits. Functional assessment of select rBDs included computational modeling as well as in vitro antimicrobial and cell migration assays. Results & Conclusion - Exposure to high-fat diet feeding for a period of three weeks resulted in a multifold-increase in cardiac mRNA expression of select rBDs, while short-term LPS exposure resulted in a smaller, but statistically non-significant, elevation in the myocardial expression of rBDs. Synthetic analogues of two naturally occurring cardiac rBDs were evaluated for in vitro activity. The synthetic rBD11 peptide exhibited antimicrobial activity against Staph aureus, and both rBDs exhibited chemoattraction of rat leukocytes. Our data suggests that rBDs might play a central role in the intrinsic immune mechanisms of the cardiovasuclar system, and possibly act as protectors of heart health.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / The enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections.

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.