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57 matches in the database. These are records: 1 - 30.

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1. Andreatta-Van Leyen, Sheila. Experimental approaches for enhancing wound healing and inhibiting tumor growth.

Degree: PhD, Physiology and Biophysics, 1994, Case Western Reserve University

► Growth factors are regulatory peptides involved in many processes of cell physiology… (more)

▼ Growth factors are regulatory peptides involved in many processes of cell physiology and pathology, including tissue repair and tumor growth. Part of this work concerned the development of a genetically engineered biological bandage (GEBB) designed to improve wound healing. Human epidermal keratinocytes (SCC-13) were genetically engineered to produce bovine growth hormone (bGH), giving raise to higher producer bGH cell lines (1 ug/ 1 × 106 cells/24hs). Selection of biocompatible polymers for bandage composition and appropriate conditions for cell maintenance was also established. The GEBB consists of a hydrophylic and gas permeable top and bottom membranes (Z-bind) united by a gasket. The bottom membrane allows cell attachment and growth, and diffusion of macromolecules. This system was functional under ambient conditions; when placed onto a full-thickness, surgically generated wound on rats, the cells within the bandage released bGH for three days. The GEBB represents a versatile system for delivering a variety of biological agents to wounds. In the second part of these studies, the role of retinoic acid (RA) and epidermal growth factor (EGF) in the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) was addressed. IGFBP-3 regulates insulin-like gr owth factors (IGFs) which in turn are important modulators of epithelium proliferation. Treatment of the ectocervical epithelial cell line ECE16-1 with EGF caused a marked reduction in IGFBP-3 levels. In contrast, RA increased mRNA and protein levels in the presence or absence of EGF. This response was concentration-dependent with a half-maximal increase observed at 1 nM RA. RA was able to reverse the EGF supression in IGFBP-3 when added simultaneously or three days after EGF treatment. Conversely, when ECE16-1 were treated with RA, IGFBP-3 levels increased within 24 h and subsequent addition of EGF was without effect. Thus, the RA-dependent increase in IGFBP-3 is dominant over the EGF supression. Increased IGFBP-3 levels correlated with supressed growth. RA also increased IGFBP-3 mRNA in other ectocervical cell lines. These results suggest that RA may act to inhibit ectocervical proliferation by increasing IGFBP-3 levels and/or reducing the extracellular concentration of free IGFs Advisors/Committee Members: Eckert, Richard L.

Subjects: Biology, Animal Physiology

Keywords: Wound healing; Tumor inhibiting; Experimental approaches

2. Beach, Jordan R. Roles and Regulation of Nonmuscle Myosin II During Cytokinesis and Epithelial-Mesenchymal Transition.

Degree: PhD, Physiology and Biophysics, 2011, Case Western Reserve University

► Nonmuscle myosin II plays an important yet incompletely understood role in many… (more)

▼ Nonmuscle myosin II plays an important yet incompletely understood role in many settings, including cytokinesis, cell migration and cell invasion. The goal of this research was to further characterize myosin II regulation and more precisely define the role that myosin II plays during these processes. In vitro studies show that RLC phosphorylation at Ser1/Ser2/Thr9 has inhibitory effects on myosin II assembly/activation. The significance of this phosphorylation in live cells has not been well studied. We show that while these sites are dramatically phosphorylated throughout mitosis, including in the contractile ring of dividing cells, that this phosphorylation has no effect on myosin II furrow recruitment or assembly levels. Spatially-restricted phosphorylation of RLC on Thr18/Ser19 via the centralsplindlin-RhoA signaling pathway has been proposed as the dominant model for myosin II recruitment to the furrow. Using a myosin II construct with a deleted RLC binding site we show that de novo filament assembly, mediated through RLC phosphorylation at Thr18/Ser19, is not the exclusive mechanism for myosin II furrow recruitment. Using truncation analysis, we provide evidence that as yet unknown binding partners recruit myosin II to the furrow through interactions with the rod domain. We show that myosin II isoforms IIA and IIB can co-assemble in the contractile ring, and that this co-assembly is dependent on bipolar filament formation. Finally, despite functional significance of myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGFbeta signaling is unknown. TGFbeta induction of EMT in mammary epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA. These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGFbeta signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state. Abrogating myosin IIB expression has no effect on 2D migration but significantly reduced transmigration and invasion. These studies suggest that transitions between myosin IIB/IIC expression and possibly MHC IIA phosphorylation are events that contribute to basal cell invasiveness. Taken together, this thesis work advances our understanding of myosin II roles and regulation during cytokinesis, migration and invasion. Advisors/Committee Members: Egelhoff, Tom.

Subjects: Cellular Biology

3. Beidelschies, Michelle. Orthopaedic Wear Particle-Induced Activation of Macrophages.

Degree: PhD, Physiology and Biophysics, 2009, Case Western Reserve University

► Osteolysis adjacent to implant surfaces is initiated by implant-derived wear particles that… (more)

▼ Osteolysis adjacent to implant surfaces is initiated by implant-derived wear particles that activate macrophages to release pro-inflammatory cytokines. This response promotes the differentiation of osteoclast precursors into mature osteoclasts. Substantial osteolysis results in implant loosening and the need for revision surgery. The presence of adherent endotoxin on the surface of wear particles increases these biological responses. Thus, the overall goal of this dissertation was to identify the cellular mechanisms utilized by adherent endotoxin on the surface of wear particles to activate macrophages.Chapter 2 examines the effects of adherent endotoxin on wear particle-induced activation of the MAPK and NF-κB pathways, and their role in TNFα production. Wear particles with adherent endotoxin induce substantial TNFα production, and strong MAPK and NF-κB activation compared to “endotoxin-free” wear particles. ERK1/2 pathway inhibitors block wear particle-induced TNFα production whereas p38 and JNK pathway inhibitors have indistinguishable effects compared to their relatively inactive analogues. Expression of Egr-1, a transcription factor downstream of ERK1/2, is stimulated by wear particles and potentiated by adherent endotoxin. Additionally, TNFα promoter activation induced by wear particles is blocked when the Egr-1 or NF-κB site is deleted or mutated. These findings demonstrate that adherent endotoxin contributes to the biological activity of wear particles through activation of the ERK1/2/Egr-1 and NF-κB pathways. Chapter 3 examines the contribution of endotoxins, other than LPS, to TNFα production induced by wear particles. Also, the requirement of adherent endotoxin for TLR-dependent TNFα production induced by wear particles was determined. TNFα expression is reduced in macrophages lacking TLR-2, the primary receptor for Gram-positive endotoxins, following stimulation with wear particles with adherent endotoxin. Moreover, both LTA and LPS adhere to and increase the biological activity of wear particles through activation of TLR-2 and TLR-4, respectively. Polymyxin B, a peptide antibiotic that inhibits the biological activity of LPS, is ineffective at blocking TNFα production induced by wear particles with adherent endotoxin and Heat-killed E. coli. However, it completely blocks TNFα production induced by wear particles that have LPS added-back and soluble LPS. These findings demonstrate that various endotoxins contribute to TNFα production and are required for a TLR-dependent response. Advisors/Committee Members: Smith, Corey.

Subjects: Biomedical research; Cellular biology; Immunology

Keywords: Wear particle; macrophage; cytokine; signaling; Toll-like receptor

4. Blum, Andrew E. G Protein-Coupled Receptor Regulation of ATP release from Astrocytes.

Degree: PhD, Physiology and Biophysics, 2010, Case Western Reserve University

► Extracellular nucleotides contribute to a complex autocrine / paracrine signaling network in… (more)

▼ Extracellular nucleotides contribute to a complex autocrine / paracrine signaling network in most tissues by activating members of the P2 receptor family. While stimulated ATP release has been demonstrated in a variety of mammalian cells, how ATP is released remains poorly understood. This dissertation illustrates the ability of G protein-Coupled Receptor (GPCR) activation to potentiate an osmosensitive ATP release pathway from 1321N1 human astrocytoma cells.Activation of the GPCRs protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs in 1321N1 human astrocytoma cells elicits rapid ATP release that depends on intracellular Ca2+ mobilization and Rho-family GTPase signaling. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca2+ mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. GPCR-regulated and hypotonic stress mediated ATP release from 1321N1 cells exhibits regulatory and pharmacological properties of volume-sensitive organic anion channels (VSOAC) type channels. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium and was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. In contrast to PAR1-dependent ATP export, PAR1-independent ATP release triggered by strong hypotonicity requires neither Ca2+ mobilization nor Rho-GTPase activation. Thus, GPCR stimulation and hypotonicity drive separate signaling cascades that converge on an ATP release pathway. A final group of experiments assessed whether additional ATP release pathways exist in 1321N1 cells. Reduction of extracellular divalent cations, which gates the opening of connexin hemichannels, elicited ATP release from 1321N1 cells that exhibited a graded attenuation in response to reduced temperature, while the GPCR- and hypotonic stress- induced ATP release responses were insensitive to similar temperature reductions. This indicated the presence of multiple ATP release pathways in 1321N1 cells. In summary, ATP release is a common response to GPCR activation, osmotic stress, and gating of non-junctional connexin channels in astrocytes that permits integration of local environmental stresses to mediate homeostatic functions. Advisors/Committee Members: Dubyak, George.

Subjects: Cellular biology

Keywords: ATP release, 1321n1, osmoregulation, VRAC, VSOAC, VSOR, Ca2+, calcium, Rho, Rho-GTPase, astrocyte, astrocytes, hypertonic, hypotonic

5. Breckenridge, Mark T. Cell Biological and Microfabrication Approaches Towards the Understanding of Transmigration and Nonmuscle Myosin II Assembly.

Degree: PhD, Physiology and Biophysics, 2010, Case Western Reserve University

► To better understand mammalian nonmuscle myosin II (NM-II) assembly regulation, mutant NM-IIA… (more)

▼ To better understand mammalian nonmuscle myosin II (NM-II) assembly regulation, mutant NM-IIA constructs were created to analyze the live cell consequences of disrupting two widely studied models of filament assembly control. A GFP-NM-IIA construct missing the RLC binding domain (ΔIQ2) destabilizes the 10 S sequestered monomer state and a GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) prevents tailpiece based regulation of assembly. Both mutants cause NM-IIA overassembly, but the GFP-NM-IIAΔIQ2 results in a severe defect in monomer recycling from assembled filaments to the leading edge during spreading, and from the cell posterior to the leading edge during polarized migration. A GFP-NM-IIA construct carrying a point mutation changing the single mapped phosphorylation site in the tailpiece from a serine to an alanine (S1943A) recapitulates the Δtailpiece phenotype. The S1943A tailpiece mutant validates the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6 S/10 S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with human MYH9-related disorder. To study the roles of nonmuscle myosin II (NM-II) during invasive cell migration (transmigration), a novel microfluidic migration chamber (the transpore chamber) has been designed and fabricated using photo- and soft-lithography microfabrication techniques. The chamber utilizes pores of graded dimension to impose differential mechanical resistance to transmigration. The pores are transverse to two convective flow channels in which two solutions of different chemoattractant concentration are flowed to generate a gradient within the pores to bias cell migration. To demonstrate the efficacy of the chamber, we compared breast-cancer cell chemotactic transmigration with and without inhibition of NM-II. Cells appeared to migrate at two distinct rates, and the faster rate was critically dependent on NM-II motor activity. The transpore chamber provides novel information on how pore size affects cell morphology and migration rate by allowing live cell monitoring of chemotaxis through pores of different dimensions within a single experimental system, providing a dramatic improvement relative to other in vitro transmigration systems. Advisors/Committee Members: Hopfer, Ulrich.

Subjects: Biology; Biomedical research; Cellular biology

Keywords: Nonmuscle myosin II; NM-II; microfabrication; chemotaxis; myosin assembly

6. Brister, Aaron B. OASIS AND XBP-1 ACTIVITY IN OSTEOBLAST DIFFERENTIATION AND OSTEOSARCOMA.

Degree: MS, Physiology and Biophysics, 2008, Case Western Reserve University

► The Unfolded Protein Response (UPR) within the Endoplasmic Reticulum (ER) is a… (more)

Keywords: XBP-1; GRP78; OASIS; OSTEOBLAST; OASIS AND XBP-1; Protein; UPR

7. Chess, David J. The Role of Dietary Fat and Carbohydrate in Cardiac Hypertrophy and Failure.

Degree: PhD, Physiology and Biophysics, 2009, Case Western Reserve University

► Despite advanced diagnostic techniques, heart failure (HF) remains a clinical, societal, and… (more)

▼ Despite advanced diagnostic techniques, heart failure (HF) remains a clinical, societal, and economic burden. Pharmacological treatments effectively manage HF but increase medical care cost by prolonging survival without improving prognosis. Thus, novel therapies that act independently of medicinal targets are needed to prevent or slow the progression of HF. Diet, a major component of daily life, is a potential strategy to impact HF pathogenesis. Thus, the overall goal of this dissertation was to assess the direct effects of dietary macronutrients on the myocardium during pathological cardiac hypertrophy and failure. The main objectives were (i) to assess the effects of carbohydrate composition on the development of hypertrophy and contractile dysfunction and (ii) compare high carbohydrate/low fat diets with low carbohydrate/high fat diets on HF pathogenesis. Studies were performed in the well-established transverse aortic constriction (TAC) model of chronic pressure overload in mice. In the first series of studies, mice were subjected to aortic banding or sham surgery and placed on either a control, high starch, or high fructose diet for 16 weeks. Animals on the high fructose diet exhibited increased mortality, left ventricular (LV) hypertrophy and remodeling, systolic dysfunction, impaired mitochondrial enzyme activities, and dramatic upregulation of myosin heavy chain β (MHCβ) compared to starch-fed mice. Treatment with the antioxidant tempol attenuated the deleterious effects of fructose, consistent with the idea that high sugar diets increase myocardial oxidative stress. Evidence of a role for insulin signaling in the adverse effects of fructose was inconclusive, as addressed in cardiac insulin receptor knockout (CIRKO) mice. The second series of studies compared high fat to high carbohydrate diets on the hypertrophic response. Following both short- and long-term dietary treatment, fat-fed animals had comparable hypertrophy and dysfunction compared to high carbohydrate-fed mice. Associated with high fat feeding was preserved mitochondrial enzyme activities, suggesting that increased fat consumption may preserve mitochondrial function and normal cardiac energetics. These findings suggest that dietary macronutrient composition can have a major impact on HF development and progression. The pleiotropic actions of diet, in conjunction with targeted drug therapies, may suppress the incidence and slow the progression of chronic disease. Advisors/Committee Members: Stanley, William.

Keywords: TAC; sham; Diet; FAT; Fructose; HYPERTROPHY; Starch

8. Choo, Chee-Keong. Human papillomavirus type 16 E6 and E7 oncogene expression in relation to host cell growth and differentiation.

Degree: PhD, Physiology and Biophysics, 1994, Case Western Reserve University

► In chapter II and III, we examined the effects of extracellular calcium… (more)

▼ In chapter II and III, we examined the effects of extracellular calcium regulation of cervical cell differentiation and HPV16 E6/E7 expression in response to calcium induced differentiation in a HPV16 immortalized cervical cell line, ECE16-1, and a HPV16-positive cervical carcinoma cell line, CaSki. ECE 16-1 and CaSki cells respond to increased calcium with increased levels of mRNA encoding differentiation markers(K13, involucrin and TG). The overall differentiation marker expression in CaSki cells is lower than in ECE16-1 cells. Protein levels change directly with the change in their corresponding mRNA, suggesting that mRNA level determines protein concentration. Transcription runoff experiments on ECE 16-1 cells indicate that the increased mRNA does not result from new synthesis. Parallel studies in ECE16-1 and CaSki cells indicate that expression of the HPV16 E6/E7 oncogene is not regulated by extracellular calcium induced differentiation, suggesting that differentiation-independent expression of E6/E7 levels is a property of both tumorigenic (CaSki) and non-tumorigenic (ECE16-1) HPV16-positive cervical epithelial cells. In chapter IV we evaluate the effects of all-trans retinoic acid (RA) on growth, dif ferentiation, expression of c-myc, and expression of HPV E6/E7 in HPV16 immortalized cervical cells. Our results indicate that the differentiation markers are suppressed by retinoids in HPV16 immortalized cervical cell lines. They are more sensitive to the effects of retinoic acid than normal cells. The level of HPV16 transcript encoding E6/E7 is not significantly suppressed by 1 uM RA in ECE 16-1 cells, but is suppressed in ECE16-D1 and ECE16-D2 cells. In addition, a transient rise in HPV transcripts encoding E6/E7 is observed at intermediate (10 and 100 nM) RA concentrations in ECE16-1 and ECE16-D2 cells. c-myc mRNA levels are elevated (5.5-fold) in HPV16-immortalized cells relative to normal cells, and are not regulated by RA. RA does not regulate proliferation in ECE16-D1 or ECE16-D2 cells but slightly suppress ECE16-1 cell growth. The levels of E6/E7 can be regulated independent of cell growth, suggesting that E6/E7 mRNA levels can be regulated independent of effects on cell proliferation in confluent cultures. In chapter V, we demonstrate that the responses of the cells fall into four groups. In the first group, E6/E7 RNA level is directly correlated with proliferation rate and inversely correlated with p53 level. In group two, E6/E7 level is directly correlated with proliferation rate, but p53 levels are not regulated. In group three, p53 level is inversely correlated with proliferation rate, but E6/E7 RNA level is not regulated. In the fourth group there is no regulation of E6/E7 level, p53 level or proliferation. RB protein levels were also assayed and found not to be regulated by RA, IFNγ or TGFβ1 in any of the cell lines. These results indicate that the response of cells to agents that regulate cell differentiation and proliferation may not always be mediated via effects on p53 and/or E6/E7 RNA level and suggest that other pathways may be as important in some circumstances. (Abstract shortened by UMI. Advisors/Committee Members: Eckert, Richard L.

Keywords: Human papillomavirus 16 E6 E7 oncogene expression relation host cell growth differentiation

9. Choowongkomon, Kiattawee. Structure and Interactions of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor.

Degree: PhD, Physiology and Biophysics, 2005, Case Western Reserve University

► The epidermal growth factor receptor (EGFR) is a member of the receptor… (more)

Subjects: Biophysics, General

Keywords: EGFR, NMR, juxtamembrane, structure, interaction, micelles, RDC

10. Christopher, Bridgette A. The Influence of Myofilament Protein Modification and Myocardial Insulin Resistance on Pathologic Left Ventricular Function.

Degree: PhD, Physiology and Biophysics, 2011, Case Western Reserve University

► In the first study, a rat model of low myocardial blood flow… (more)

▼ In the first study, a rat model of low myocardial blood flow tested whether post-translational changes to proteins of the thin and thick muscle filaments correlate with decreased cardiac contractility. Following three days of low blood flow, cardiac trabeculae demonstrated a reduction in fractional shortening at rest and a relative decline in fractional shortening when challenged with high dose dobutamine, reflecting reduced energy reserves. Permeabilized fibers from low blood flow hearts demonstrated a decline in maximum force and Ca2+ sensitivity. An examination of sarcomeric proteins by two-dimensional gel electrophoresis, mass spectrometry, and phospho-specific antibodies showed Ser23/24 and Ser43/45 phosphorylation of troponin I (TnI). Total TnI phosphorylation was not different between the groups, but Ser23/24 phosphorylation declined with low blood flow, implying an accompanying increase in Ser43/45 phosphorylation of TnI. These findings suggest that altered TnI function, due to changes in the distribution of phosphorylated sites, is an early contributor to reduced contractility of the heart. Previous studies have reported that high fat feeding in mild to moderate heart failure (HF) results in the preservation of contractile function, suggesting that preventing the switch from fatty acid to glucose metabolism in HF may ameliorate dysfunction. Insulin resistance is one potential mechanism for regulating substrate utilization, and the second study sought to determine whether peripheral and myocardial insulin resistance exists with HF and/or high fat diet. Rats underwent coronary artery ligation (HF) or sham surgery and were randomized to normal chow (NC) or high fat diet (SAT) for eight weeks. HF+SAT showed preserved systolic and diastolic function compared to HF+NC. Glucose tolerance tests revealed peripheral insulin resistance in SHAM+SAT, HF+NC, and HF+SAT compared to SHAM+NC. Positron emission tomography imaging confirmed myocardial insulin resistance only in HF+SAT. Western blot analysis of insulin signaling protein expression was indicative of cardiac insulin resistance in HF+SAT. In conclusion, HF animals fed high saturated fat exhibit preserved myocardial contractile function, peripheral and myocardial insulin resistance, decreased myocardial glucose utilization rate and alterations in cardiac insulin signaling. These results suggest that myocardial insulin resistance may serve a cardioprotective function with high fat feeding in mild to moderate heart failure. Advisors/Committee Members: Chandler, Margaret.

Subjects: Physiology

Keywords: TnI; myocardial infarction; high saturated fat; myocardial insulin resistance

11. Cianciola, Nicholas L. Homeostasis of Endocytic and Autophagic Systems: Insights from the Host-Pathogen Interaction.

Degree: PhD, Physiology and Biophysics, 2010, Case Western Reserve University

► The host-pathogen interaction provides a unique system offering insight into both the… (more)

▼ The host-pathogen interaction provides a unique system offering insight into both the mechanisms by which pathogens subvert host cellular processes, and the biological processes themselves. The aim of this study was to understand the molecular basis of action of the adenovirus protein RIDα, which was originally identified by its ability to divert constitutively recycling EGF receptors to lysosomes during an adenovirus infection, and also when expressed in cells devoid of other adenovirus proteins. We report that RIDα usurps host cell machinery to exit the trans-Golgi network by an AP-1-dependent mechanism. A tyrosine motif located in the carboxyl tail of RIDα mediates this interaction, and mutation to alanine blocks biosynthetic export and inhibits RIDa function. We also found that RIDα mimics GTP-Rab7 by binding two Rab7 effector proteins, RILP and ORP1L, which coordinately recruit the motor proteins necessary for movement of late endosomes along microtubules. As RIDα shares no sequence homology with Rab7 and has no intrinsic enzymatic activity, we asked how RIDα is regulated to mimic Rab7. It was discovered that RIDα is post-translationally modified by the addition of the lipid palmitate to the lone cysteine residue in the carboxyl tail.Mutation of this site did not affect the localization of RIDα, as both wild-type and mutant proteins localized to a unique perinuclear “autophagy-like” compartment. However, palmitoylation does affect protein function, as expression of the RIDα palmitoylation-deficient mutant altered the morphology of LAMP1-positive late endosomes. These enlarged compartments were loaded with cholesterol and other lipids, similar to the phenotype observed in the cholesterol storage disease Niemann-Pick type C (NPC). While mutant RIDα disrupted sterol regulated gene regulation, wild-type RIDα was found to rescue the defects created by the mutations that cause NPC disease. This novel function of RIDα was found to be dependent on the class III phosphatidylinositol 3-kinase that regulates the expansion of autophagosomal membranes. We conclude that RIDα activates an autonomous cholesterol egress mechanism that is independent of the NPC disease gene products. As a model system, RIDα provides insight into the coordination and homeostasis of endocytic and autophagic systems, and the role of cholesterol in these pathways. Advisors/Committee Members: Smith, Corey.

Subjects: Biology; Microbiology; Molecular biology

Keywords: adenovirus; endocytosis; palmitoylation; autophagy; cholesterol homeostasis

12. Coley, Austin A. Immunocytochemical techniques identify Na+-coupled HCO3– transporters (NCBTs) in chemosensitive neurons of the medullary raphé.

Degree: MS, Physiology and Biophysics, 2011, Case Western Reserve University

► The medullary raphé (MR) of the medulla oblongata contains chemosensitive (serotonin/5-Hydroxytryptamine [5HT],… (more)

Subjects: Biophysics; Neurosciences; Physiology

Keywords: nNCBTs, NBCn1, NBCn2, NDCBE, Serotonergic neurons, GABAergic neurons, pHi, Immunocytochemistry, Image processing

13. Conway, James Patrick. Systems Biology Analysis of Macrophage Foam Cells: Finding a Novel Function for Peroxiredoxin I.

Degree: PhD, Physiology and Biophysics, 2007, Case Western Reserve University

► Atherosclerosis is a type of cardiovascular disease that is characterized by localized… (more)

▼ Atherosclerosis is a type of cardiovascular disease that is characterized by localized thickening of the arteries at sites referred to as atheromas. The earliest events leading to atheroma formation involve the oxidative modification of low-density lipoprotein in the arterial intima, and the uptake of oxidatively-modified low-density lipoprotein by intimal macrophages. This unregulated uptake results in lipid-laden macrophage-derived foam cells, which aggregate at potential sites of atheroma development known as fatty streaks. We hypothesized that the transition from macrophage to foam cell is characterized by differential expression that, as a whole, is pro-atherogenic. A proteomic and transcriptomic analysis identified several proteins and mRNA transcripts that were differentially-regulated following acute and chronic exposure to oxLDL, including mediators of antioxidant and immune response. Further, chronic oxLDL exposure led to a decrease in oxLDL-induced toxicity, which coincided with an increased antioxidant response when compared to macrophages treated with a single exposure to oxLDL. Data obtained from the analysis of differential expression in foam cells provided a foundation for the second stage of this project. Peroxiredoxin I was identified as a protein that is upregulated in macrophages following exposure to oxLDL, which led to the hypothesis that the upregulation of peroxiredoxin I could protect against toxicity induced by oxLDL-generated reactive oxygen species. It was determined that peroxiredoxin I can decrease oxLDL-induced toxicity when induced prior to oxLDL-exposure. This effect coincides with a decrease in reactive oxygen species, verifying the antioxidant role of peroxiredoxin I. Additionally, alternative functionality for peroxiredoxin I was tested, and it was determined that the activation of p38 MAPK is dependent on peroxiredoxin I expression. The sum of this data suggests that peroxiredoxin I contributes to cell survival and signaling, which affects macrophage foam cell function and survival. Advisors/Committee Members: Kinter, Mike T.

Subjects: Biology, Cell

Keywords: atherosclerosis; foam cell; macrophage; oxLDL; peroxiredoxin; p38 MAPK; proteomics

14. Crumrine, Ralph Christian. Protein kinase C activity, metabolic, and hemodynamic changes in cerebral ischemia in the adult rat.

Degree: PhD, Physiology and Biophysics, 1990, Case Western Reserve University

► Cerebral high energy metabolites and metabolic end products were measured during and… (more)

▼ Cerebral high energy metabolites and metabolic end products were measured during and following total cerebral ischemia in the rat (TCI). During ischemia, lactate accumulation was greatest in the hippocampus, followed by the cerebral cortex and striatum. Following reperfusion, the rate of lactate clearance was slower in the hippocampus than in the other two regions. Regional cerebral blood flow (CBF), cerebral plasma volume (CPV) and calculated mean transit time (MTT) were determined following reflow of ischemic tissue. During hyperemia, CPV increased concomitantly with CBF while the MTT remained near control, suggesting that the linear flow rate through the vasculature was unchanged. During hypoperfusion, CPV returned to control values; but, there was a significant increase in MTT. The finding of normal tissue energy charge, pH i, and concentration of other metabolites during hypoperfusion suggest that hypoperfusion does not result in CBF-metabolic mismatch and implies cerebral hypometabolism during this period. Protein kinase C (PKC) activity during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was use d as an index of PKC activation. There was a drop in percent particulate PKC activity after 30 minutes of ischemia. There was a 40% decline of the total cellular PKC activity after 20 minutes of TCI. This was not accompanied by an increase in activator-independent activity, indicating PKC was not being converted to protein kinase M. These data suggest that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 hours of recovery following 11-13 minutes of TCI. There was a reduced level of total, soluble and particulate PKC activity in the cerebral cortex corresponding to the decrease observed by 15 minutes of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator-independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and this reduced level of PKC activity was maintained throughout 6 hours of recovery. Pathologic activation of PKC was not associated with ischemic damage. Advisors/Committee Members: LaManna, Joseph C.

Keywords: kinase C metabolic hemodynamic cerebral ischemia adult rat

15. Doreian, Bryan William. Molecular Regulation of the Exocytic Mode in Adrenal Chromaffin Cells.

Degree: PhD, Physiology and Biophysics, 2009, Case Western Reserve University

► Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and… (more)

▼ Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release while elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. We show that at low frequency stimulation mimicking sympathetic tone, a filamentous-actin cell cortex plays a role in stabilizing the kiss-and-run fusion event. Increased stimulation, mimicking input under the acute stress response, disrupts the actin cortex, driving full granule collapse. We show pharmacological perturbation of the actin cortex supersedes stimulus frequency in controlling exocytic mode. Our data demonstrate that phosphorylation of MARCKS under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phosphorylated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity dependent exocytosis of the peptide transmitters. Lastly, it was found that disruption of dynamin I function alters both modes of exocytosis and that its dephosphorylation is a critical step for this transition in exocytic mode. Under low stimulation, dynamin I remains phosphorylated. Interference with dynamin I did not affect granule fusion but did block its re-internalization. Under high stimulation, dynamin I is dephosphorylated, and can bind syndapin/N-WASP to promote focal actin polymerization. Inhibition of syndapin binding or N-WASP activity limited fusion pore dilation. Furthermore, in the full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. Taken together, these data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore, promoting the binding of syndapin and associated molecules for focal actin polymerization, and thus contributing to activity-dependent differential transmitter release from the adrenal medulla. Advisors/Committee Members: Smith, Corey.

Subjects: Biology; Biophysics

Keywords: Chromaffin, Exocytosis, Myosin II, MARCKS, F-actin

16. Etwebi, Zienab. Magnesium Regulation of Glucose and Fatty Acid Metabolism in HEPG2 Cells.

Degree: MS, Physiology and Biophysics, 2011, Case Western Reserve University

► Magnesium (Mg2+) is an important cation for a variety of cell functions… (more)

Subjects: Physiology

17. Falin, Rebecca A. Acute Inhibition of the Epithelial Sodium Channel.

Degree: PhD, Physiology and Biophysics, 2008, Case Western Reserve University

► The epithelial sodium channel (ENaC) is expressed in a variety of tissues,… (more)

▼ The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. First, the ERK1/2-mediated down-regulation of ENaC was characterized in primary and immortalized renal collecting duct cells (mCT12). Addition of epidermal growth factor (EGF) to polarized monolayers reduced amiloride-sensitive short-circuit current (ISC) by 15-25%. Exposure of mCT12 cells to EGF caused an increase in phosphorylation of ERK1/2; pretreatment of monolayers with a MEK inhibitor prevented this phosphorylation and significantly reduced EGF-induced inhibition of amiloride-sensitive ISC. The results of these studies demonstrate that acute inhibition of ISC by EGF involves ERK1/2 activation. p>Liddle's syndrome is caused by gain-of-function mutations in the beta- and gamma-subunits of ENaC, resulting in enhanced Na+ reabsorption and hypertension. In the second part of this study the effect of EGF on sodium absorption in primary renal collecting duct cells derived from a Liddle's mouse model was evaluated. It was found that EGF inhibited ISC by 24+/-5% in wild-type cells but only by 6+/-3% in homozygous mutant cells. EGF-induced ERK1/2 phosphorylation was similar in +/+ and L/L cells, and prolonged exposure to EGF decreased ENaC expression by ~50% in both. Acute inhibition of ENaC activity by EGF requires an intact beta-ENaC C-terminus, whereas EGF-induced down-regulation of ENaC expression does not. Finally, in order to elucidate the role of specific regions of the beta-ENaC C-terminus, MDCK cell lines expressing beta-ENaC with mutations of the PY motif, ERK phosphorylation site, and C-terminus truncation were created. All mutants exhibited significant attenuation of EGF-induced inhibition of sodium current. In MDCK cells with wild-type beta-ENaC, EGF-induced inhibition of ISC (<30min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (Po). At later times (>30min), EGF-induced inhibition of ISC was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. These results are consistent with a reversible ERK-mediated decrease in ENaC Po followed by an irreversible retrieval of sodium channels from the apical membrane. Advisors/Committee Members: Cotton, Calvin U.

Keywords: ENaC beta; Epidermal Growth Factor; Ubiquitin; ERK MAP Kinases; Kidney Collecting Duct

18. Giannattasio, Bartolomeo. Characterization of ATP receptors and voltage-dependent calcium ion channels in cardiovascular cells.

Degree: PhD, Physiology and Biophysics, 1993, Case Western Reserve University

► The primary goal of this study was to characterize myocardial ATP receptors.… (more)

Subjects: Biology, Animal Physiology

Keywords: ATP receptors; Calcium channels; Cardiovascular cells

19. Gorodeski, George Israel. Retinoids and steroid hormones regulate differentiation of cultured human ectocervical cells.

Degree: PhD, Physiology and Biophysics, 1990, Case Western Reserve University

► The present study was predicated on the hypothesis that retinoids (analogues of… (more)

▼ The present study was predicated on the hypothesis that retinoids (analogues of vitamin A) have a direct effect on human female reproductive tract epithelia and that they modulate the effects of estradiol (E2) and progesterone (P) on these tissues. To study retinoid and steroid regulation of differentiation in vitro I used cultured ectocervical epithelial cells (ECE) grown on 3T3 monolayers. Confluent ECE cells free of non-epithelial cells as judged by lack of expression of vimentin, stratified spontaneously, terminally differentiated and formed cornified envelopes. The epithelial origin of these cells was determined by their morphology and the expression of protein and mRNA involucrin and cytokeratins. Differentiation of the ECE cells could be modulated in vitro by diethylstilbestrol (DES) and P and the cells expressed specific E2 binding sites (receptor). Four experiments were done: (1) Retinoids (Ro 13-6298, trans Retinoic Acid, Retinol) modulate differentiation of ECE cells in concentrations of 10-11M to 10-7M resulting in loss of stratification, suppression of envelopes (mainly Ro 13-6298) and increase in cell number (mainly Retinoic Acid). (2) Steroids, in concentrations of 10-11M to 10-6M also modulate differentiation: DES and Hydrocortisone (HC) increase while P and Testosterone suppress envelopes formation and HC increases cell number. Experiments using combinations of retinoids and steroids revealed that retinoids are more potent suppressors of envelopes than P, that DES and retinoids synergistically increase cell number and that DES renders the cells sensitive to P suppression of envelopes. (3) Changes in involucrin protein and mRNA are not the basis for envelopes regulation by retinoids and steroids. (4) Retinoids modulate cytokeratins expression: K13 (and mRNA) and K16 levels decrease and K19 (and mRNA) levels increase. Based on these results I conclude that in-vivo: (1) Vitamin A determines the basal degree of differentiation of ectocervical cells. (2) The sex steroid hormones regulate the state of differentiation of the ectocervical tissues around this set point. (3) Vitamin A regulates squamous metaplasia of the squamo-columnar junction by increasing the fraction of reserve cells. (4) Vitamin A maintains the upper reproductive tract epithelium in the form of simple columnar epithelium. Advisors/Committee Members: Eckert, Richard L.

Keywords: Retinoids; steroid hormones; cultured; ectocervical cells

20. Gorski, David Henry. Homeobox gene expression and regulation in vascular myocytes.

Degree: PhD, Physiology and Biophysics, 1994, Case Western Reserve University

► This dissertation examines the potential roles of homeobox genes in growth regulation… (more)

▼ This dissertation examines the potential roles of homeobox genes in growth regulation in vascular smooth muscle cells, a topic whose interest derives from its relevance to disease processes in which vascular myocyte proliferation is disordered, such as atherosclerosis. Abnormal proliferation of vascular smooth muscle cells is a critical component of the pathogenesis of both of these conditions, and very little is known about transcription factors which might play a role in regulating the entry of these cells into the cell cycle. To determine if homeobox transcription factors are involved in this process, a vascular cDNA library was screened with a degenerate oligonucleotide probe directed at the most conserved region of the homeodomain. Several homeobox cDNAs were isolated, including one that encodes Gax, a diverged homeobox gene that was subsequently found to be rapidly down-regulated when quiescent vascular smooth muscle cells were stimulated with serum growth factors. Further studies revealed another, previously described, homeobox gene which is responsive to serum growth factors, MHox/phox, whose transcript is rapidly up-regulated upon mitogen stimulation. Because of its unique pattern of expression, we tested recombinant Gax protein for its ability to inhibit the entry of vascular smooth muscle cells into S-phase. Gax inhibited the entry of quiescent vascular myocytes into S-phase by 40%. We compared this to MyoD and an anti-ras antibody, which inhibited vascular myocyte entry into S-phase by 36% and 65%, respectively. The growth inhibitory effect of microinjected Gax was dose-dependent and reversed by highly oncogenic ras mutant, Ras (Leu-61). Time course microinjection experiments suggested that Gax acts somewhere in late G1 to inhibit vascular myocyte proliferation. These experiments suggest that Gax is a negative regulator of growth in vascular myocytes, and that it is likely that Gax and other homeobox genes play a role in regulating the modulation of vascular myocytes from the contractile to the synthetic phenotype. Advisors/Committee Members: Walsh, Kenneth.

Keywords: Homeobox gene expression regulation vascular myocytes

21. Hill, Jacqueline Suzanne. "Mechanisms of Adrenal Medullary Excitation Under the Acute Sympathetic Stress Response".

Degree: PhD, Physiology and Biophysics, 2012, Case Western Reserve University

► Regulation of systemic catecholamine levels is critical for maintenance of integral physiological… (more)

▼ Regulation of systemic catecholamine levels is critical for maintenance of integral physiological functions such as heart rate, insulin secretion and regulation of blood flow within the body. The adrenal medulla, the innermost layer of the adrenal gland, contains neurosecretory chromaffin cells. Chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnic-released acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympatho-adrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. Sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, Pituitary Adenylate Cyclase-Activating Peptide (PACAP), which acts through a mechanistically distinct pathway from cholinergic excitation. In this study, I investigate the cellular and tissue-level mechanisms through which PACAP evokes excitation and sustained secretion. I report that PACAP stimulates sustained catecholamine release through a PKC-dependent functional recruitment of nickel-sensitive T-type Cav3.2 channels. To investigate this stimulus path, I utilized perforated- patch clamp electrophysiology, immunohistochemistry, and carbon fiber amperometry. I show that despite abundant immunohistochemical staining for Cav3.2, there is little nickel-sensitive current present in the unstressed adrenal medulla. Subsequently, I show that PACAP-dependent PKC activation, functionally recruits latent Cav3.2 channels to a functional, current-producing form. The functional recruitment of Cav3.2 generates a tonic Ca2+ window current to support sustained secretion. Moreover, previous studies showed that chronic stress results in an electrical remodeling of the adrenal medulla by up-regulation of connexin protein expression. Other studies report that phospho-regulation of existing gap junction channels acts to increase junctional conductance. I tested the hypothesis that PACAP-mediated excitation up-regulates cell-cell electrical coupling to enhance chromaffin cell excitability. I utilized electrophysiological recordings to measure the effects of PACAP stimulation on cell coupling. I report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. These findings show that PACAP acts as a secretagogue and remodels electrical remodeling of the medulla to adapt to the organism’s needs during acute sympathetic stress. Advisors/Committee Members: Smith, Corey.

Subjects: Neurosciences; Physiology

Keywords: Acute stress, voltage-gated calcium channels, PACAP, gap junctions, patch clamp electrophysiology

22. Hong, Shiyuan. Expression and Function of ART2.1 ecto-ADP-ribosyltransferase in Inflammatory Effector Cells.

Degree: PhD, Physiology and Biophysics, 2009, Case Western Reserve University

► The ATP-gated P2X7 purinergic receptor (P2X7R) is predominantly expressed in cells of… (more)

▼ The ATP-gated P2X7 purinergic receptor (P2X7R) is predominantly expressed in cells of hematopoietic origin including macrophages and T lymphocytes. Roles for the P2X7R have been identified in the regulation of various pro-inflammatory and immune responses. An unusual feature of the P2X7R is its high threshold for activation by extracellular ATP (EC50 ~ 500 μM); this contrasts with much lower activation thresholds for the other six members of the P2X family (EC50 ~ 10 μM). Recent studies have demonstrated that extracellular NAD induces the ATP-independent activation of the P2X7R in murine T lymphocytes via ADP-ribosylation of arginine residues on the P2X7R ecto-domain. This modification is catalyzed by ART2.2, a GPI-anchored ADP¬ribosyltransferase (ART) that is constitutively expressed in murine T cells. Although the NAD-induced, ART2-dependent mechanism is clearly a major pathway for P2X7R activation in mouse T lymphocytes, it is unclear whether this mechanism is operative in macrophages, another class of leukocytes which natively express P2X7R at high levels. The studies described in this dissertation addressed this question in following two folds. The first series of studies (Chapter 3) demonstrated that bone-marrow derived macrophages (BMDM) from BALB/c mice selectively up-regulate thiol-dependent ART2.1 in response to multiple proinflammatory mediators including agonists for toll-like receptors (TLR) and type-1/2 interferons. LPS-induced ART2.1 expression involved regulation by multiple kinase pathways or possibly IFN-β autocrine signaling. A related group of studies (Chapter 5) showed expression of ART2.1 in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid antigen-prensenting leukocytes. In contrast with T cells, stimulation of naïve or inflammatory macrophages with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation in inflammatory macrophages as indicated by a left-shift in the ATP dose-response relationship (Chapter 4). These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in murine macrophages and that the cellular context in which P2X7R signaling occurs differs between myeloid versus lymphoid leukocytes. Collectively, this dissertation demonstrated that the regulation of ATP-dependent P2X7R activation by NAD/ART2.1 provides an additional layer of regulatory control in multiple phases of innate and adaptive immunity. Advisors/Committee Members: Dubyak, George R.

Subjects: Cellular biology

Keywords: P2X7 receptor ecto-ADP-ribosyltransferase ATP NAD leukocyte lymphocyte macrophage LPS Interferon

23. Iancu, Radu Vlad. cAMP COMPARTMENTATION IN ADULT CARDIAC MYOCYTES.

Degree: PhD, Physiology and Biophysics, 2008, Case Western Reserve University

► Receptor-mediated changes in cAMP production play an essential role in hormone regulation… (more)

▼ Receptor-mediated changes in cAMP production play an essential role in hormone regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In the present study, we used a systems biology approach to investigate the role played by cAMP compartmentation in cardiac ventricular myocytes and the mechanisms underlying it.In the first part of this study, we used a computational approach to test the hypothesis that in ventricular myocytes the effects of β1-adrenergic receptor (β1AR) and M2 muscarinic receptor (M2R) activation involve compartmentation of cAMP. Results obtained with the developed model indicate that (1) bulk basal cAMP can be high (~1 μM) and only modestly stimulated by β1AR activation (~2 μM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A - PKA (~100 nM to ~2 μM); (2) M2R activation strongly reduces the β1AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and (3) during weak β1AR stimulation, M2R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP. The original model suggests that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the second part of this study we experimentally tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer (FRET)-based biosensor constructed from the type 2 exchange protein activated by cAMP. Our results support the conclusion that even though β1 and M2 receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. In the final part of the current study, we investigated the potential mechanisms responsible for cAMP compartmentation. We conclude that both limited cAMP diffusion between the submembrane and bulk compartments and an increased level of phosphodiesterase (PDE) activity in the membrane compartment are necessary for generating and maintaining the type of cAMP gradients previously characterized using intracellular FRET based cAMP probes. Advisors/Committee Members: Harvey, Robert.

Subjects: Biophysics

Keywords: cAMP, myocyte, computer model, cAMP compartments

24. Jacobs, Leila Susan. Role of sphingolipids in regulation of vascular smooth muscle-derived A7r5 cell proliferation.

Degree: PhD, Physiology and Biophysics, 1993, Case Western Reserve University

► The role of sphingolipids in mediating the action of platelet-derived growth factor… (more)

▼ The role of sphingolipids in mediating the action of platelet-derived growth factor (PDGF) has been investigated in the vascular smooth muscle-derived A7r5 cell line. L-cycloserine (2 mM), an inhibitor of sphingolipid synthesis, caused time-dependent inhibition of (3H) serine incorporation into (3H) sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml) or sphingosine (10 uM) independently stimulated (3H) thymidine incorporation into DNA of A7r5 cells. L-cycloserine (2 mM) inhibited stimulation of DNA synthesis by both PDGF-AB and PDGF-BB. L-cycloserine (2 mM, 16 h) did not affect the ability of PDGF or sphingosine to increase intracellular free calcium ( (Ca2+) i) in A7r5 cells loaded with the fluorescent indicator fura-2. Measurement of adenine nucleotide levels in A7r5 cell extracts by reverse-phase high-performance liquid chromatography indicated that treatment with L-cycloserine did not adversely affect cellular metabolism. To determine directly whether PDGF activates sphingolipid metabolism, A7r5 cells were labeled with (3H) serine for 48 h and then treated with PDGF-AB (10 ng/ml) for 1 h. Sphingolipids were separated by thin-layer chromatography and quantified by liquid scintillation counting. PDGF-AB stimulated an increase in (3H) sphingosine from 25.5 ± 3.0 to 37.5 ± 4.1 cpm/ug protein and a concomitant decrease in (3H) ceramide from 24.3 ± 3.2 to 18.5 ± 2.9 cpm/ug protein. Exogenous sphingosine (10 uM, 6 min) stimulated an increase in (32P) lysophosphatidic acid but had no effect on (32P) lysophosphatidylcholine, suggesting specificity of sphingosine action. In A7r5 cells labeled with 1-O- (3H) alkyl-2-lyso-sn-glycero-3-phosphocholine, exogenous sphingosine stimulated a dose-dependent increase in (3H) phosphatidic acid. Exogenous sphingosine produced a dose-dependent increase in (32P) sphingosine-1-phosphate, indicating that A7r5 cells have an active sphingosine kinase. These data suggest that the PDGF-stimulated increase in (Ca2+) i is not sufficient for induction of DNA synthesis and that mitogenic effects of PDGF in vascular smooth muscle cells are mediated in part by sphingolipid metabolism. Advisors/Committee Members: Kester, Mark.

Subjects: Biology, Cell

Keywords: sphingolipids regulation vascular smooth muscle-derived A7r5 cell proliferation

25. Kim, Jane H. The WT1 Interacting Protein: a choreographer of podocyte morphology and transcription.

Degree: PhD, Physiology and Biophysics, 2011, Case Western Reserve University

► Podocyte structural and transcriptional phenotypic plasticity characterizes glomerular injury. Activity of the… (more)

▼ Podocyte structural and transcriptional phenotypic plasticity characterizes glomerular injury. Activity of the transcription factor Wilm’s Tumor-1 (WT1) is required for normal podocyte structure and is repressed by the podocyte adherens junction protein, WT1 Interacting Protein (WTIP). We show that WTIP translocated into podocyte nuclei in lipopolysaccharide (LPS)-treated mice and cultured podocytes, a model of transient nephrotic syndrome. LPS-stimulated WTIP nuclear translocation required JNK activity, which assembled a multiprotein complex of the scaffolding protein JIP3 and the molecular motor dynein. Intact microtubule (MT) networks and dynein activity were necessary for LPS-stimulated WTIP translocation. Stress signaling pathways initiate WTIP nuclear translocation, suggesting a mechanism that transmits changes in podocyte morphology to the nucleus. Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. WTIP, an Ajuba family Lin11, Isl-1 and Mec-3 (LIM) domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in isolated adherent podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Non-contacted shWtip podocytes did not assemble actin stress fibers and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and B-catenin clustered in irregularly distributed spots that failed to laterally expand. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined if Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor, C3 toxin, and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix requires Wtip, a process that may be mediated by spatio-temporal regulation of RhoA activity through appropriate targeting of Arhgef12. Advisors/Committee Members: Smith, Corey.

Subjects: Molecular Biology

Keywords: WTIP; podocyte; kidney; actin

26. King, Kristen L. The Role of Mitochondrial Thioesterase-I, Uncoupling Protein-3, and CD36 in Cardiac Mitochondria.

Degree: PhD, Physiology and Biophysics, 2008, Case Western Reserve University

► Cardiac dysfunction and cardiomyopathy are associated with excessive accumulation of lipids in… (more)

▼ Cardiac dysfunction and cardiomyopathy are associated with excessive accumulation of lipids in the heart. This condition can be mimicked by cardiac specific overexpression of peroxisome proliferator-activated receptor α (PPARα). Mitochondrial thioesterase I (MTE-I), a mitochondrial matrix protein whose expression is controlled by PPARα, de-esterifies long chain acyl-CoAs into coenzyme A and fatty acid anions (FA-), thus it may play a role in reducing mitochondrial accumulation of toxic long chain acyl-CoAs and preventing lipotoxic cardiomyopathy. It is unclear how the generated FA- leave the mitochondrial matrix, but two mitochondrial proteins capable of exporting FA- have recently been identified: uncoupling protein 3 (UCP3) and fatty acid translocase (FAT) CD36. While it has been shown that diabetes or pharmacological activation of PPARα up-regulate mRNA expression of MTE-I and UCP3, little is known about the effects on protein expression or FA- export from mitochondria. Thus the goal of this dissertation is to elucidate the roles of MTE-I, UCP3, and CD36 in cardiac mitochondria, specifically examining the effects of diabetes and PPARα stimulation. First we studied the effects of treatment with the PPARα agonist fenofibrate or streptozotocin-induced diabetes on MTE-I and UCP3 protein expression and activity in isolated cardiac mitochondria in rats. Both diabetes and fenofibrate increased cardiac 9 MTE-I mRNA, protein and activity approximately 4-fold compared to controls. UCP3 protein expression was not increased despite a 4-fold increase in mRNA. Palmitate export from mitochondria increased 6-fold in fenofibrate treated animals. Both diabetes and fenofibrate significantly decreased state III respiration of isolated mitochondria with pyruvate+malate as the substrate, but only diabetes reduced state III rates with palmitoylcarnitine. While the mechanism for this effect remains unclear, it appears that the decreased cardiac mechanical efficiency and increased myocardial oxygen consumption seen in diabetes is not related to changes in MTE-I activity or FA- transport. Secondly, we studied the role of another fatty acid transporter, FAT/CD36, in cardiac and skeletal muscle mitochondria. Pharmacological studies suggest that CD36 may play an essential role in mitochondrial fatty acid oxidation. We further postulated that CD36 transports FA- out of the mitochondrial matrix. We assessed mitochondrial respiration and palmitate export in isolated cardiac mitochondria from wild type and CD36 knock-out mice. Despite the clear presence of CD36 in cardiac mitochondria from WT mice, the absence of CD36 did not affect palmitoylcarnitine oxidation, or the export of FA- generated in the matrix. Thus CD36 does not play an essential role in mitochondrial uptake of fatty acids or export of FA-. Advisors/Committee Members: Stanley, William C.

Subjects: Biology, Animal Physiology

Keywords: Mitochondrial Thioesterase-I; Uncoupling Protein-3; CD36; Cardiac Mitochondria

27. Kuri, Barbara Alison. REGULATION OF CATECHOLAMINE RELEASE FROM THE ADRENAL MEDULLA UNDER THE PHYSIOLOGICAL STRESS RESPONSE.

Degree: PhD, Physiology and Biophysics, 2010, Case Western Reserve University

► Release of catecholamine from the adrenal glands is a highly regulated process.… (more)

▼ Release of catecholamine from the adrenal glands is a highly regulated process. The chromaffin cells of the adrenal medulla are the parenchymal cells of the adrenal gland and are capable of releasing catecholamine (adrenaline and noradrenaline) and neuroactive peptides into the circulation. Under autonomic nervous control, the innervating splanchnic nerve directs the release of catecholamine. In this study, we show how chronic intermittent hypoxia (CIH), a common form of metabolic stress, affects the release of catecholamine from the adrenal chromaffin cells, in situ. Using electrophysiological capacitance recording techniques we show that exposure to CIH results in an increase of the amount of catecholamine available for release, upon a given stimulus, through the recruitment of catecholamine containing granules to the readily releasable pool. We show that this is possible through the generation of reactive oxygen species (ROS) and subsequent activation of protein kinase C (PKC). To further address the regulation of catecholamine release under sympathetic stimulation we developed a bipolar stimulation protocol that mimics stimulation frequencies at rest and under burst mode stress firing. Using an in situ adrenal gland slice preparation we show that there is an activity dependent regulation of catecholamine release involving pituitary adenylate cyclase activating peptide (PACAP) which is released from the splanchnic nerve under conditions mimicking the burst mode stress firing. We show that PACAP acts through a novel mechanistic pathway that is independent of the conventional sympathetic cholinergic pathway. This characteristic of PACAP makes it an ideal neurotransmitter under high intensity, burst mode sympathetic firing, in that it does not desensitize like the cholinergic pathway. We show that PACAP acts through a G-protein coupled receptor to activate phospholipase C (PLC) which in turn activates PKC. Activation of PKC plays a role in increasing the activity of the sodium calcium exchanger (NCX) in the forward direction to depolarize the cell membrane to potentials that increase the open probability of a Ni2+ sensitive, low voltage activated Ca2+ channel. It is the influx of Ca2+ through this channel to generate the Ca2+ dependent release of catecholamine. Advisors/Committee Members: Smith, Corey.

Subjects: Anatomy and physiology; Biology; Biophysics; Cellular biology

Keywords: Chromaffin, Stress, Catecholamine, Hypoxia, Amperometry

28. Kusner, David John. Regulation of phospholipase D activity in U937 cells.

Degree: PhD, Physiology and Biophysics, 1994, Case Western Reserve University

► The goal of this project was to study the regulation of phospholipase… (more)

▼ The goal of this project was to study the regulation of phospholipase D (PLD) activity in the human promonocytic U937 cell line. We have studied activation of PLD by GTPγS, and its potentiation by ATP, in a cell-free system derived from U937 promonocytic leukocytes. ATP, in the micromolar to millimolar range, significantly augmented GTPγS-stimulated PLD activity (2.6-fold) and the combination resulted in a 15-fold increase in PLD activity compared to control. ATP alone did not stimulate PLD activity. Measurement of endogenous cytosolic ATP levels and nucleotide depletion of cytosol with activated charcoal demonstrated that stimulation of PLD by GTPγS proceeds by both ATP-dependent and ATP-independent pathways. Nucleotide specificity data suggested that the ATP-dependent pathway involves kinase activity. Tyrosine kinase activity is neither necessary nor sufficient for activation of PLD in this system. We have analyzed the effect of G-protein activation on the subcellular requirements for reconstitution of PLD activity using the cell-free system from U937 cells. Incubation of cells with 100 uM GTPγS, in the presence of the permeabilizing agent β-escin, followed by isolation of plasma membranes, resulted in a membrane-localized PLD activity which was indep endent of added cytosol. The PLD activity of plasma membranes from GTPγS-treated cells was 7-fold greater than the basal activity of membranes isolated from cells incubated with β-escin alone, and could be further augmented by the addition of 1 mM ATP. Cytosol from GTPγS-treated cells had a decreased capacity to support PLD activation (when combined with control membranes) consistent with G-protein-induced depletion of a factor essential for reconstitution of PLD activity. Treatment of cells with other compounds known to activate PLD (PMA, vanadate) did not result in a stable membrane-localized PLD activity. Incubation of isolated plasma membrane and cytosol with GTPγS, followed by reisolation of the membranes, also resulted in a cytosol-independent PLD-activity in the membranes. While Ca+2 was not required for this G-protein-mediated effect, micromolar Ca+2 potentiated the GTPγS-dependent association of PLD activity with the reisolated membranes. The third part of this project involved analysis of the cytosolic factor required for reconstitution of G-protein-dependent PLD activity. Preliminary characterization of the cytosolic factor was performed by ammonium sulfate fractionation, and chromatography on hydrophobic, anion-exchange, and nucleotide affinity columns. As each of these techniques yielded a single major peak of PLD-reconstituting activity, it is proposed that only one cytosolic factor is required. Further enrichment of the cytosolic factor by sequential fractionation should permit more detailed characterization of the regulation of PLD in phagocytic leukocytes. Advisors/Committee Members: Dubyak, George R.

Keywords: Regulation of phospholipase D activity in U937 cells.

29. Lashin, Ossama M. Functional modification of cardiac mitochondria in type-I diabetes.

Degree: PhD, Physiology and Biophysics, 2005, Case Western Reserve University

► Diabetes is a strong risk factor for several chronic diseases and for… (more)

▼ Diabetes is a strong risk factor for several chronic diseases and for premature mortality. Among other complications, the diabetic patient develops cardiomyopathy due to several metabolic, biochemical and ultrastructural changes involving the cardiac tissue. This myocardial dysfunction not only is considered one of the most serious and life threatening factors, but also worsens the prognosis of the diabetic patient in the event of other cardiac insults such as ischemia and myocardial infarction. Numerous studies have shown that diabetic cardiomyopathy involves several functional alterations within the cardiac myocyte. In this respect, mitochondria have received a great deal of attention as they represent the main cellular source of energy. The decline in the respiratory function of mitochondria from diabetic tissues has been mainly attributed to defects in several enzymes of the mitochondrial electron transport chain. However, the presence and the extent of these defects are controversial due to several factors, including the different diabetic animal models used, the parameters considered in classifying animals as diabetic or not, and tissues utilized to isolate mitochondria. In addition, the extent of the reported defects in electron transport in diabetic mitochondria is not sufficient to explain per se the differences in respiration observed between diabetic and non diabetic mitochondria. A unifying factor between diabetes and mitochondrial dysfunction is the existence of cellular oxidative damage. Several studies have shown that diabetes is in fact a state of oxidative stress. The mechanisms by which oxidative stress occurs and the generation of reactive oxygen species increases under diabetic conditions are not fully elucidated. Because mitochondria are one of the main cellular sources of ROS, it is believed that mitochondrial dysfunction in diabetic tissues largely contributes to the increased oxidative damage present in diabetic tissues. One of the main consequences of oxidative damage is the production of lipid peroxidation products, including aldehydes, from phospholipids of the biological membranes as a result of reactions involving free radicals. The generated lipid peroxidation products are highly toxic to the cells and can cause structural and functional modifications to cellular proteins. Based on these considerations we hypothesize that the existent mitochondrial defects in diabetic hearts, specifically in the electron transport chain, is a major source of ROS. In turn, ROS can react with and modify unsaturated lipids from the mitochondrial membrane, thus generating the lipid peroxidation product HNE, which can further react with key mitochondrial proteins or enzymes. This sequence of events can represent an important mechanism by which mitochondrial respiratory function is altered in diabetic hearts and contribute to the development of diabetic cardiomyopathy. To test this hypothesis, we used a streptozotocin diabetic rat model to address the following specific aims: 1) To examine the mitochondrial oxygen consumption as an index of mitochondrial respiratory function over a time course of 12 weeks of diabetes; 2) To determine the development of oxidative stress in diabetic mitochondria by detecting HNE modified mitochondrial proteins; 3) To identify specific HNE-modified mitochondrial proteins, and determine the functional consequence of their modification; and 4) To ascertain whether this process can be prevented or reversed by insulin administration. The elucidation of these main points can help to understand the nature of the defect(s) in mitochondria function in diabetic hearts, and the consequence of oxidative damage on key mitochondrial proteins. Advisors/Committee Members: Romani, Andrea.

Keywords: Diabetes; Mitochondria; Reactive oxygen species; Lipid peroxidation; Protein modification

30. Leygerman, Milana. The Effects of Cardiac Myosin Binding Protein-C and Inorganic Phosphate on Length-Dependent Activation.

Degree: MS, Physiology and Biophysics, 2011, Case Western Reserve University

► The contractile unit of a striated muscle is called the sarcomere and… (more)

▼ The contractile unit of a striated muscle is called the sarcomere and is composed of actin, myosin, titin, the troponin complex, tropomyosin, the myosin light chains, and cardiac myosin binding protein-C (cMyBP-C). Muscle contraction is caused by cross-bridge cycling, which involves the sliding of thick filaments past thin filaments. cMyBP-C is a constituent of the thick filament and is involved in regulation of contraction. Mutations in this protein have been known to lead to hypertrophic cardiomyopathy, an autosomal disease characterized by hypertrophy and fibrosis. Sarcomere length is an important determinant of muscle contractility as increased length results in greater overlap between actin and myosin leading to greater Ca2+-sensitivity and force generation. The Frank-Starling law of the heart is an important relationship for regulation of cardiac muscle contraction and is influenced by sarcomere length. In conditions of heart failure, there is a downward and rightward shift of the Frank-Starling relationship where an increase in end-diastolic volume generates a relatively smaller increase in stroke volume insufficient to meet the demands of the heart. Inorganic phosphate plays an important role in muscle contraction as its release from the acto-myosin complex is a crucial step in the cross-bridge cycle that drives muscle contraction. In this study, to elucidate the effects of cMyBP-C and inorganic phosphate on length dependent activation, we utilized a skinned myocardium isolated from a knockout (KO) mouse model that lacks cMyBP-C (cMyBP-C-/-). A total of 85 mechanical experiments were performed in skinned fibers isolated from WT and KO left ventricles. The results showed that increased sarcomere length increases force generation and Ca2+-sensitivity of force in WT and KO animals. The rate of force development, an index of the speed of cross-bridge function was accelerated with increased sarcomere length in the KO fibers but not WT fibers. Treatment of muscle fibers with a low concentration of inorganic phosphate (1mM) did not have an effect on maximum calcium-activated force at short and long sarcomere length in either WT or KO myocardium, but accelerated rates of force development in both WT and KO muscle fibers. These results imply that the effects of sarcomere length are enhanced in KO myocardium due to the increased proximity of myosin to actin in fibers lacking cMyBP-C. Low concentrations of inorganic phosphate may accelerate the transitions from weak to strong binding cross-bridge states in both WT and KO myocardium, thereby accelerating rates of force development. Advisors/Committee Members: Stelzer, Julian.

Subjects: Physiology

Keywords: Cardiac myosin binding protein-C, inorganic phosphate, length-dependent activation, cross-bridge cycling

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