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 Anatomy and Physiology / A. Philine Wangemann / Pendred syndrome is characterized by sensorineural deafness and post-pubertal goiter. It is caused by mutations in the anion exchanger, pendrin (SLC26A4). The purpose of this study was to understand the etiology of Pendred syndrome using a mouse model. Different methods of amplification from nanogram amounts of starting RNA were evaluated for gene array application. Gene arrays were performed and free radical stress markers were compared between the stria vascularis and the thyroid of the Slc26a4+/- and Slc26a4-/- mice. Hearing loss in Slc26a4-/- mice is linked to the loss of Kcnj10 protein expression and consequently the loss of endocochlear potential. To understand the mechanism of hearing loss in Slc26a4-/- mice, progressive loss of Kcnj10 protein expression in stria vascularis of Slc26a4-/- mice was assessed, the modulation of Kcnj10 protein expression by free radical stress in cultured stria vascularis and in an heterologous expression system was evaluated. To characterize the thyroid pathology, pendrin expression in the thyroid of Slc26a4+/- mice was measured in a developmental study and correlated with serum thyroxine (T4) levels of Slc26a4+/- and Slc26a4-/- mice over a developmental time course. All tested methods of RNA amplification were suitable for gene array application and demonstrated high internal consistency. Intermethod comparisons revealed variations in data, suggesting that a single amplification method ought to be used within a given experiment. Markers of free radical stress were increased in the stria vascularis of Slc26a4-/- mice before the onset of hearing. Progressive loss of Kcnj10 expression was seen in Slc26a4-/- mice at the onset of hearing. Furthermore, free radical stress modulated the expression of Kcnj10 in cultured stria vascularis and in a heterologous expression system. The pendrin mRNA expression was marginal in the thyroid and did not correlate with serum T4 levels. Further, absence of pendrin did not affect free radical stress markers in the thyroid. These data suggest that free radical stress-mediated loss of Kcnj10 expression in stria vascularis contributes to deafness in the Pendred syndrome mouse model and that pendrin is not essential for the function of mouse thyroid gland.

Master of Science / Department of Kinesiology / Timothy I. Musch / Dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) containing docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) has been demonstrated to produce advantageous effects on vascular function. Specifically, PUFA supplementation has resulted in enhanced brachial artery blood flow (Q), dilation, and vascular conductance (VC) during rhythmic handgrip exercise. The effects of fish oils (FO) on skeletal muscle blood flow (Qm) during dynamic whole body exercise, however, remain unknown. PURPOSE: To test our hypothesis that 6 weeks of dietary FO supplementation with DHA and EPA enhances regional Qm and VC to the hindlimb musculature during submaximal treadmill exercise. METHODS: Following 6 weeks of dietary supplementation with safflower oil (SO) (control; n = 9) or FO (n = 8), heart rate (HR), mean arterial pressure (MAP), and Q[subscript]m to the hindlimb were measured at rest and during submaximal treadmill exercise (20 m/min, 10%, ~65% VO[subscript]2max) via radiolabeled microspheres in adult male Sprague-Dawley rats. RESULTS: HR and MAP were not different between SO and FO at rest or exercise (P<0.05). Q[subscript]m and VC were not different between SO and FO at rest. During exercise, FO exhibited greater Q[subscript]m in 8 of the 28 muscle parts measured as well as greater VC in 11 of the 28 muscle parts measured. Additionally, FO exhibited greater (Q)[subscript] m (158[plus or minus]9) and VC (1.156[plus or minus]0.066) to the total hindlimb musculature than SO (128[plus or minus]10 ml/min/100g, 0.918[plus or minus]0.077 ml/min/100g/mmHg) (P<0.05). CONCLUSION: These results demonstrate that 6 weeks of dietary FO supplementation with DHA and EPA results in enhanced Q[subscript]m and VC to the hindlimb during submaximal exercise. Thus, supplementation with DHA and EPA may have therapeutic effects on oxygen delivery and vascular function in patients with impaired vascular function and exercise tolerance (i.e., congestive heart failure, diabetes).

Master of Science / Department of Kinesiology / Timothy I. Musch / Advancing age alters the structural and functional determinants of convective and diffusive muscle oxygen (O[subscript]2) flux. However, capillary red blood cell (RBC) hemodynamics have not been investigated during contractions in muscles of old animals. PURPOSE: To test the hypothesis that aging induces significant alterations in capillary hemodynamics during electrically-induced contractions in the spinotrapezius muscle of old Fischer 344 x Brown Norway rats when compared to younger counterparts. METHODS: The spinotrapezius muscle was observed via intravital microscopy in 8 old (O: 26-30 months) and 5 young (Y: 6-8 months) animals. Wire electrodes elicited 1 Hz (6-8 volts) contractions for 3 minutes. RBC flux (FRBC), velocity (VRBC), capillary hematocrit (HCAP), and total microvascular O[subscript]2 delivery (QO[subscript]2m) were measured both at rest and during the steady-state of muscle contractions. RESULTS: At rest F[subscript]RBC and V[subscript]RBC were elevated in O compared to Y rats, while there was no difference in HCAP or QO[subscript]2m between groups. During the contracting steady-state, [Delta]F[subscript]RBC (Y: 28.8 [plus or minus] 7.7, O: -2.9 [plus or minus] 1.4 cells/s), [Delta]V[subscript]RBC (Y: 253 [plus or minus] 68, O: -4 [plus or minus] 15 [Mu]m/s), [Delta]H[subscript]CAP (Y: 0.02 [plus or minus] 0.02, O: -0.03 [plus or minus] 0.01 cells/[Mu]m), and [Delta]QO[subscript]2m (Y: 892 [plus or minus] 255, O: -24 [plus or minus] 30 cells/s/mm) cells/s/mm were all lower (P < 0.05) in O compared to Y rats. CONCLUSION: These results indicate that despite maintained total convective and diffusive O[subscript]2 transport at rest, advancing age results in significant alterations in capillary hemodynamics during electrically-induced contractions. These alterations likely contribute to the mechanisms responsible for the reduced exercise capacity commonly found in elderly populations.

Master of Science / Department of Animal Sciences and Industry / Barry J. Bradford / The prevalence of fatty liver in transition dairy cattle has been reported to be as high as 50%. There are a few reliable on-farm diagnostic tools and even fewer methods to effectively prevent fatty liver. Non-alcoholic steatohepatitis, an advanced form of non-alcoholic fatty liver in humans, is accurately diagnosed with a commercial blood test that detects plasma cytokeratin-18 (CK18) fragments released during hepatocyte apoptosis. A study was performed using 89 Holstein cows in early lactation to determine if CK18 could serve as a novel indicator of liver triglyceride (TG) content. Although no previous work has been done with CK18 in bovine plasma, our results indicated that CK18 fragments were present in plasma. However, CK18 concentrations did not correlate with liver TG content or other measures of liver function, suggesting it is not a reliable diagnostic tool. Nevertheless, based on liver TG, plasma non-esterified fatty acid (NEFA), and plasma β-hydroxybutyric acid (BHBA) concentrations, this sample population as a whole was not suffering from severe metabolic problems or fatty liver, making it possible that plasma CK18 fragments are elevated only in the most extreme cases. Currently, there is no widely-adopted preventative strategy for fatty liver. A second study was performed to evaluate if encapsulated niacin (EN) could prevent liver TG accumulation during the transition period. Twenty-four primiparous (n=9) and multiparous (n=13) cows were randomly assigned to receive 0 or 24 g of dietary EN, beginning 3 weeks prior to expected calving until 21 days postpartum. Feeding EN did not influence liver TG content, but decreased plasma NEFA concentrations, suggesting inhibition of lipolysis. Multiparous EN cows also experienced depressed dry matter intake (DMI) in the 4 days prior to calving. However, even when EN reduced DMI, plasma NEFA was still suppressed. A novel finding was the prolonged clearance of caffeine in plasma on day 7 postpartum in EN-treated animals. In contrast to other studies, this dose and delivery method of EN did not result in an increase in plasma NEFA after EN treatment ended. These research projects determined that plasma CK18 is likely not a useful diagnostic tool for mild to moderate bovine fatty liver and that feeding EN can inhibit lipolysis but may influence DMI as well. This is one of the first studies into the metabolic effects of feeding EN, and further research is needed in this field.

Master of Science / Department of Animal Sciences and Industry / James S. Drouillard / Christopher D. Reinhardt / The effects of supplementing forms of flaxseed on plasma and longissimus muscle (LM) fatty acid (FA) composition, finishing performance, and carcass characteristics were evaluated in five studies. In study 1, steers were fed diets with soy oil (SO), ground flaxseed (Flaxseed), or urea formaldehyde condensation polymer treated flaxseed (UFCP). In study 2, steers were fed diets with SO, linseed oil (LO), or a combination of flaxseed and field peas that was extruded (LinPro). Feeding flaxseed products increased (P < 0.01) α-linolenic acid (ALA), omega-3 FA, and decreased (P < 0.01) n-6:n-3 in LM compared to cattle fed SO. Feeding LinPro increased (P < 0.01) ALA, omega-3 FA, and decreased (P < 0.01) n-6:n-3 in LM compared to steers fed SO or LO. In studies 3 and 4, steers were fed diets with and without Flaxseed and implanted or not. Implanting improved (P ≤ 0.05) DMI, ADG, feed efficiency, HCW, and LM area compared to cattle not implanted. In study 4, cattle fed Flaxseed had increased (P < 0.01) ALA and omega-3 FA, and decreased (P < 0.01) n-6:n-3 in LM compared to cattle fed SO. In study 5, heifers were fed diets with 0% or 5% linseed meal, and administered with or without exogenous hormones (NHTC). Administering exogenous hormones improved (P ≤ 0.02) DMI, ADG, G:F, and HCW compared to NHTC cattle. Omega-3 FA increased in LM when cattle were supplemented with flaxseed products. Cattle fed LinPro achieved the highest levels of ALA and omega-3 FA. Flaxseed products did not interact with implants as a natural growth promoter in finishing cattle.

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.

Master of Science / Department of Animal Sciences and Industry / KC Olson / Condensed tannins (CT) in plants are phenolic compounds with relatively high binding affinities for proteins. In ruminants, dietary CT limit DM intake and digestibility, and ruminal protein degradation by forming CT-protein complexes. Effects of dietary CT, animal species, prior dietary CT exposure, and antimicrobial inclusion on 48-h rate and extent of digestion were measured in two in vitro experiments. Cattle, sheep, and goats (n = 3 / species) were used in a 2-period, randomized complete-block experiment with a 2 × 3 × 2 × 3 factorial arrangement of treatments. Factor 1 was substrate: tannin-free or high-CT. Factor 2 was source of ruminal fluid inoculum: cattle, sheep, or goat. Factor 3 was prior animal exposure to a high-CT diet: non-exposed or exposed. Factor 4 was inclusion of antimicrobials: no antimicrobial, penicillin + streptomycin to suppress bacterial activity, or cycloheximide to suppress fungal activity in the fermentation. Tannin-free or high-CT substrates were incubated in vitro using ruminal fluid from animals either not exposed (period 1) or exposed to dietary CT (period 2). Periods consisted of an adaptation to tannin-free (10 d) or high-CT diets (21 d) and a 15-d period of ruminal-fluid collection via stomach tube. The presence of CT or penicillin + streptomycin in in vitro fermentation reduced (P < 0.001) total gas pressure, DM disappearance, and total VFA, acetate, propionate, butyrate, valerate, and branched-chain VFA concentrations. We concluded that: 1) CT had negative effects on fermentation, 2) prior exposure to dietary CT attenuated some but not all negative effects, and 3) CT effects were similar to the effects of penicillin + streptomycin.

Master of Science / Department of Entomology / Yoonseong Park / Neuropeptides are diffusible signal molecules mediating vital physiological processes. We have been interested in a group of neuropeptides and their receptors involved in osmoregulatory neuroendocrine system which has been suggested as a possible target for development of new biopesticides. Since the genome sequence of the T. castaneum has recently been completed, we were able to identify the respective genes encoding three peptide hormones from T. castaneum that were characterized for their diuretic activities in other insects: one calcitonin-like (CT-like DH31) and two corticotropin releasing factor-like (CRF-like DH37 and DH47, the numbers indicates the number of amino acid residues). This peptide is expressed at all developmental stages and in the central nervous system (CNS), Malpighian tubules (MT) and gut. The synthetic peptide TricaDH31 also has been show to be biologically active, inducing significant excretions in adults beetles. When Tcdh31 was silenced using RNAi, adults had deformed wings and abnormal body shape. Mortality in adults was high, the number of eggs laid was reduced as well as the hatchability of the eggs. The two biologically active CRF-like peptides in T. castaneum, are encoded by one gene which undergoes alternative splicing. When Tcdh47 was knocked down, high mortality occurred as well as low oviposition and egg hatchability. Similar effects were observed with silencing of both CRF-like genes. However, RNAi of Tcdh37 transcripts had similar, but less severe effects. Adults also had deformed wings when both CRF-like genes were silenced, but not when just one of them was knocked down. These results indicate that CRF-like genes could have additional biological functions to their roles in dieresis. We tested the in vivo activity of these peptides. TenmoDH47 induced high excretions in adults, whereas TenmoDH37 induces smaller excretions. We identified the respective genes encoding two putative receptors for TricaDH31 as Glean_13321 and Glean_02694 (Trica-ctr1 and Trica-ctr2, respectively) and two receptors for CRF-like peptide as Glean_12799 and Glean_07104 (Trica-crfr1 and Trica-crfr2, respectively). The CT-like receptors are expressed at all developmental stages, in the CNS and MT. RNAi of the receptors revealed that only Trica-ctr2 silencing caused significant mortality and reduction in the number of eggs laid. The CRF-like receptors are expressed at all developmental stages. Adults also had deformed wings and laid fewer eggs after RNAi of Trica-crfr1. RNAi of Trica-crf2 also caused significant mortality. These peptides and receptors seem to fine tune the beetle physiology and may have functions not yet known.