Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Thu Annelise Nguyen / Gap junctions (GJ) are intercellular channels connecting adjacent cells, allowing small molecules to transport between cells, thereby maintaining all homeostasis. Loss of gap junctional intercellular communication (GJIC) and/or connexins, the gap junction proteins, is a hallmark of cancer. Restoration of GJIC and/or increase of connexin expression have been related to the reduction of tumorigenesis. Connexins have been reported as tumor suppressors due to both GJIC-independent and -dependent mechanisms. Therefore, development of effective agents or methods to enhance GJIC and restore connexin expression in cancer cells is a new strategy in cancer treatment. PQ1, 6-Methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethyl-phenyloxy)quinoline, has been demonstrated to increase GJIC, restore connexin expression, and exert anti-cancer effects on T47D breast cancer cells. Studies of apoptotic pathways showed that PQ1 activated both extrinsic and intrinsic apoptotic pathways, indicating that PQ1 exerts its anti-cancer effects via a GJIC-independent mechanism through the induction of apoptosis. Combinational treatment of PQ1 and cisplatin showed that PQ1 counteracted cisplatin-induced inhibition of GJIC and reduction of connexin expression, thereby increasing the efficacy of cisplatin in T47D cancer cells via a GJIC-dependent mechanism. Further studies of drug distribution and toxicity revealed that administration of PQ1 by oral gavage can be achieved with low toxicity to normal vital organs. All the results suggest that PQ1, a gap junction enhancer, can function as an anti-cancer agent and potentiate the efficacy of antineoplastic drugs via both GJIC-independent and -dependent pathways.

Doctor of Philosophy / Department of Human Nutrition / Tonatiuh Melgarejo / Obesity is a metabolic disease accompanied by a disruption in the immune system leading to systemic inflammation and susceptibility to infections. Leptin, the peptide secreted by adipocytes in proportion to fat mass, is primarily a metabolic hormone translating the body’s energy status to the brain. Leptin is also a pro-inflammatory cytokine and leptin deficiency is associated with higher infection susceptibility and a protection against autoimmune diseases. Leptin’s dual metabolic-immune function places this hormone as the link between metabolic disturbances of obesity and the immune system. In the following research projects, the contribution of leptin to both inflammation and infection susceptibility was investigated in a murine model of diet-induced obesity. Chimeric mice with leptin receptor-deficient bone marrow were resistant to HFD-induced weight and fat mass gain. These mice exhibited less inflammation in the adipose tissue demonstrated by a blunted increase in tnfa and il6 gene transcript levels, a higher prevalence of anti-inflammatory macrophages and a lower number of crown-like structures. Systemically, these mice showed a tendency towards higher insulin sensitivity. These outcomes were compared to those from mice with wild-type bone marrow. Obese and lean mice exhibited similar kinetics of bacterial clearance and systemic leptin changes following infection with Ehrlichia chaffeensis in vivo. Nevertheless, isolated “obese” peritoneal macrophages were significantly less phagocytic than macrophages from lean mice and supplementation with leptin significantly increased the obese macrophages' phagocytic activity with no effect on lean macrophages. A cell line, DB-1, derived from leptin receptor-deficient bone marrow was immortalized and characterized. This cell line has phenotypic and functional properties characteristic of macrophages, lacks the long isoform of the leptin receptor, and is unresponsive to leptin. The data from the above mentioned studies suggest that leptin contributes to the inflammation of obesity. They also suggest that leptin affects macrophage function in obesity in vitro though more studies are required to assess leptin’s contribution to infection outcomes in vivo. Finally, DB-1 cells provide a dependable tool to study further the role of leptin in obesity-associated inflammation and immune system dysregulation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, a Gram-negative organism belonging to the order Rickettsiales, is responsible for an emerging infectious disease in humans, the human monocytic ehrlichiosis. E. chaffeensis also infects several other vertebrate hosts including dogs, goats, coyotes and white tailed deers. This organism is transmitted by an infected tick, Amblyomma americanum. The exact pathogenic mechanisms involved for the persistence of the pathogen in vertebrate hosts are still unclear. E. chaffeensis protein expression varies significantly in vertebrate and tick hosts. Differentially expressed proteins include the immunodominant outer membrane proteins encoded by the p28-Omp multigene locus. The p28-Omp 14 is expressed primarily in tick cells and the p28-Omp 19 is the major expressed protein in macrophages both under in vitro and in vivo conditions. The objective of this study is to prepare recombinant proteins and use them to assess the secondary structures and protein functions. The protein sequences were analyzed with the aid of bioinformatics programs to make structural predictions. The analysis suggested the presence of eight β barrel structures for both the p28-Omp proteins. The coding sequence of the p28-Omp genes were cloned and over expressions of proteins in in E. coli was accomplished by using the plasmid expression construct, pET28. The proteins were purified to near homogeneity and used to refold using detergents to mimic native protein structure in the bacterial outer membrane. Refolding of proteins was analyzed by two methods; SDS-PAGE and Circular Dichroism. The Circular dichroism spectroscopy analysis suggested the formation of β-sheet structures of proteins in micelles formed with the detergents. β-sheet structures may have been formed with the hydrophobic domains of the protein imbedded in the micelles. The hydrophilic segments (predicted by bio informatics analysis) may be exposed to the aqueous phase. The recombinant proteins were also iii used to prepare proteoliposomes and tested for the porin activity. The analysis demonstrated the porin activity for both p28-Omp 14 and 19 recombinant proteins by using mono-, di- and tetra- saccharides as well as for amino acid L-glutamine. This study forms the basis for initiating studies to compare the structural difference between the two differentially expressed proteins of E. chaffeensis.

Doctor of Philosophy / Graduate Biochemistry Group / Dolores J. Takemoto / Eukaryotic cells possess a unique way to communicate with each other by passing metabolites and small molecules through protein pores that connect adjacent cells. Although there are many types and families of protein pores, connexins comprise a unique family. Six connexin monomers assemble into a hemichannel, which is transported to the cell membrane. An opposing cell membrane containing compatible connexin hemichannels is located and connected, forming an intercellular dodecameric protein complex. This results in a protein channel that connects two separate cytoplasmic compartments to each other. This type of channel is known as a gap junction. Connexin expression and function is commonly tissue specific. Of the 21 known human connexins, less than half are currently well characterized. Three connexins are expressed in the lens, connexin 43 (Cx43), 46 (Cx46), and 50 (Cx50). Of these three, Cx46 and Cx50 both have major functions in the mature lens. Cx46 functions as a major gap junction channel, which maintains mature lens homeostasis, while Cx50 possesses growth control properties in the lens. Cx46 expression is modulated in breast and bone tumors, and during ischemia. It is hypothesized that Cx46 provides resistance to hypoxia mediated cell death by prolonging survival. In this study, Cx46 expression was detected in human Y79 retinoblastoma cells. Decreasing the expression of Cx46 in nude mice carrying Y79 xenografts slowed early stage tumor growth. Y79 cells in culture survive for over 72 hours in 1% oxygen in vitro. C46 was upregulated in cultured lens cells when grown under hypoxia. Human lens epithelial cells, rabbit N/N1003A lens cells, and Y79 cells proliferated in 1% oxygen until Cx46 expression was depleted by use of siRNA. Protection from hypoxia-induced cell death was provided by transfection with the C-terminus of Cx46. We further determined that the promoter activity of Cx46 was increased in 1% oxygen. These results indicate that Cx46 would increase in response to hypoxia and suggest a role for Cx46 in protection from hypoxia. The studies demonstrate a novel function for Cx46 in cell survival during hypoxia.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Harvey Morgan Scott / A randomized controlled field trial was designed to evaluate the effects of two treatment strategies on ceftiofur and tetracycline resistances in feedlot cattle. The two strategies consisted of administering ceftiofur crystalline-free acid administration (CCFA) at either one or else all of the steers within a pen, and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. Both strategies were hypothesized to reduce ceftiofur resistance. The effects of treatment strategies were evaluated via metagenome-based and culture-based assays. In this 26-day study, 176 steers were allocated to 16 pens of 11 steers each. The two strategies were randomly assigned to the pens in a two-way full-factorial manner resulting in four treatment groups. The blaCMY-2, blaCTX-M, tet(A), tet(B), and 16S rRNA gene copies/g feces were quantified using qRT-PCR from fecal community DNA. Antimicrobial susceptibility profiles were determined using microbroth dilution technique from the non-type-specific (NTS) E. coli isolates (n=1,050). The NTS E. coli DNA was screened for the presence of blaCMY-2, tet(A), and tet(B) genes. Pens in which all the steers received CCFA treatment showed an increase in blaCMY-2 and blaCTX-M log10 gene copies/g feces and in the proportion of ceftiofur-resistant and blaCMY-2 positive NTS E. coli. This was in contrast to the pens where only one animal received CCFA treatment. There was a significant decrease in quantities of tetracycline genes in community DNA in pens where all animals received CCFA treatment. In contrast to metagenome-based assay results, culture-based assays indicated an increase in the proportion of tetracycline resistant NTS E. coli upon CCFA treatment. Thereafter, chlortetracycline administration led to rapid expansion both of ceftiofur (blaCMY-2, blaCTX-M) and tetracycline [tet(A) and tet(B)] log10 gene copies/g feces. Chlortetracycline treatment delayed the return of the ceftiofur resistance prevalence to baseline among NTS E. coli and thus did not lead to the hypothesized decrease in ceftiofur resistance. Our data suggest that chlortetracycline use is contraindicated when attempting to avoid expansion of resistance to critically important 3rd generation cephalosporins in feedlot cattle. Further studies are required to better establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together at varying ratios on the levels of antimicrobial resistance.

Master of Science / Department of Physics / Robert Szoszkiewicz / Proteins are involved in many of the essential cellular processes, such as cell adhesion, muscle function, enzymatic activity or signaling. It has been observed that the biological function of many proteins is critically connected to their folded conformation. Thus, the studies of the process of protein folding have become one of the central questions at the intersection of biophysics and biochemistry. We propose to use the changes of the nanomechanical properties of these biomolecules as a proxy to study how the single proteins fold. In the first steps towards this goal, the work presented in this thesis is concentrated on studies of unfolding forces and pathways of one particular multidomain protein, as well as on development of the novel method to study elastic spring constant and mechanical energy dissipation factors of simple proteins and peptides. In the first part of this thesis we present the results of the mean unfolding forces of the NRR region of the Notch1 protein. Those results are obtained using force spectroscopy techniques with the atomic force microscope (AFM) on a single molecule level. We study force-induced protein unfolding patterns and relate those to the conformational transitions within the protein using available crystal structure of the Notch protein and molecular dynamics simulations. Notch is an important protein, involved in triggering leukemia and breast cancers in metazoans, i.e., animals and humans. In the second part of this thesis we develop a model to obtain quantitative measurements of the molecular stiffness and mechanical energy dissipation factors for selected simple proteins and polypeptides from the AFM force spectroscopy measurements. We have developed this model by measuring the shifts of several thermally excited resonance frequencies of atomic force microscopy cantilevers in contact with the biomolecules. Next, we provided partial experimental validation of this model using peptide films. Ultimately, our results are expected to contribute in the future to the developments of medical sciences, which are advancing at a level, where human health and disease can be traced down to molecular scale.

Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.

Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.

Doctor of Philosophy / Horticulture, Forestry, and Recreation Resources / Sunghun Park / Abiotic stresses, including chilling and drought stresses, are considered to be major limiting factors for growth and yield of agricultural and horticultural crops. One of the inevitable consequences of abiotic stresses is the accumulation of reactive oxygen species (ROS) in plants. ROS can either act as an alarm signal to induce the defense pathway when kept at a low level or cause oxidative damage to various cellular components when increased to a phytotoxic level. Glutaredoxins (GRXs) are members of ROS scavenging system that can maintain the cell redox homeostasis by using the reducing power of glutathione. In this research, we characterized the roles of GRXs in protecting tomato (Solanum lycopersicum) from chilling stresses and rice (Oryza sativa L.) from drought stresses. Our results indicated that ectopic expression of an Arabidopsis gene AtGRXS17 in tomato could enhance the chilling tolerance by increasing antioxidant enzyme activities and reducing H₂O₂ accumulation to ameliorate oxidative damage to cell membranes and photosystems. Furthermore, AtGRXS17-expressing tomato plants had increased accumulation of soluble sugars to protect plant cells from dehydration stress. In rice, silenced expression of a rice glutaredoxin gene OsGRXS17 was used as a reverse-genetic approach to elucidate the roles of OsGRXS17 in drought stress tolerance. Our results showed that silenced expression of OsGRXS17 conferred improved tolerance to drought stress in rice. ABA-mediated stomatal closure is an important protection mechanism that plants adapt to a drought stress conditions, and H2O2 acts as secondary messenger in ABA signaling to induce the stomatal closure. Silenced expression of OsGRXS17 gave rise to H₂O₂ accumulation in the guard cells and promoted ABA-mediated stomatal closure, resulting in reduced water loss, higher relative water content, and consequently enhanced drought tolerance in rice. This research provides a new perspective on the functions of GRXs in chilling and drought stress tolerance of tomato and rice, and an important genetic engineering approach to improve chilling and drought stress tolerance for other crop species.