Biomedical Engineering and Bioengineering Commons^™

Cytochrome C Oxidase From Rhodobacter Sphaeroides: Oligomeric Structure In The Phospholipid Bilayer And The Structural And Functional Effects Of A C-Terminal Truncation In Subunit Iii, Teresa L. Cvetkov

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Cytochrome c oxidase (COX) of the mitochondrial electron transport chain catalyzes the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. This two part dissertation is a structural and functional investigation of COX using the bacterial model system from Rhodobacter sphaeroides (R.sph.).

First, the oligomeric structure of R.sph. COX within the lipid bilayer was investigated using discontinuous sucrose gradient ultracentrifugation. Utilizing this technique, liposomes containing R.sph. COX (pCOV) were separated from liposomes lacking enzyme (COV). The net buffering capacity and degree of light scattering per COX molecule were reduced in pCOVs, making them well suited …

Charaterization Of Chlorpyrifos Toxicity On The Pancreatic Beta Cell Line Rinm5f, Zhongyu Yan

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Chlorpyrifos (CPF) is a well-known organophosphate (OP) insecticide that inhibits acetylcholinesterase (AChE). Organophosphates have been shown to induce hyperglycemia in both humans and animals that cannot be explained by AChE inhibition alone. Pancreatic β-cells are responsible for secreting insulin and playing a fundamental role in glucose homeostasis. The aim of this research was to investigate CPF toxicity on the insulin secreting β-cell line RINm5f (RIN). In the first part of this dissertation, we determined that cytotoxic concentrations of CPF between 50 µM and 200 µM induced a dose-dependent increase in RIN cell apoptotic and necrotic death (p<0.05). Lower doses, i.e., 5-25 µM did not induce apoptosis or necrosis. In further mechanistic studies, inhibitors of c-Jun N-terminal kinase (JNK) and P38 pathways, i.e., SP600125 and SB202190, successfully attenuated CPF-induced apoptosis (p<0.05), suggesting cell death is mediated by JNK and/or p38. In addition, N-Acetyl-L-cysteine (NAC), an antioxidant, inhibited apoptosis suggesting that oxidative stress is also involved in RIN cell death. In the second part of this dissertation, low dose CPF's effects on insulin synthesis and secretion was studied. Our results showed that RIN cells treated with CPF for 4 hr exhibited a dose-dependent decrease in insulin secretion in response to stimulation by 50 mM potassium (p<0.05). Insulin secretion stimulated by 25 mM glucose also showed significant decreases upon incubation with various doses of CPF (p<0.05). Insulin synthesis involves proteolytic cleavage of proinsulin at two specific sites. The enzymes responsible for promoting proinsulin cleavage, i.e, prohormone convertase (PC) 1/3 and 2 (PC2), contain a serine hydrolase catalytic triad, His-Asp-Ser, similar to AChE. CPF modification of PC enzymes similar to AChE, could potentially interrupt the conversion of proinsulin to insulin and disrupt insulin secretion. PC2 enzyme activity was significantly inhibited by CPF at concentrations as low as 10 µM (p<0.01). RIN cells, for the first time, were found to secrete proinsulin was through a constitutive instead of a regulated pathway. In summary, our results show that CPF not only exerts a direct cytotoxic effect at concentrations above 50 µM, but also significantly impairs insulin secretion at lower doses. Low dose CPF could potentially affect endocrine function in vivo by inhibiting the proinsulin processing enzyme PC2.

Cellular Effects Of Replicating A Polypurine-Polypyrimidine Sequence And The Interactions Of Due-B With Replication Proteins, Shere Lynne Myers

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This work investigates two questions regarding DNA replication. The first aim examines the interactions of DUE-B with replication proteins and the second explores the cellular effects of replicating a polypurine polypyrimidine sequence in human cells.

DUE-B siRNA decreases the chromatin binding of essential replication proteins Cdc45, PCNA and RPA. DUE-B also co-immunoprecipitates with Cdc45 and TopBP1. In vitro kinase assays suggest that the checkpoint protein ATR may phosphorylate DUE-B. These experiments lend further evidence that DUE-B plays an important role in the initiation of eukaryotic DNA replication.

To investigate the effects of replicating a polypurine-polypyrimidine sequence prone to secondary structure …

Differential Regulation Of Vitamin D Receptor (Vdr) By P53, P63 And P73, Ramakrishna Kommagani

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The tumor suppressor p53 is the single most altered gene in human cancers. p53 homologues, p63 and p73 play a major role in development and in human cancer. Both p63and p73-null mice exhibit profound developmental abnormalities, suggesting a vital role for p63 and p73 in development. Although the role of p73 in human cancers is well established, the role of p63 still remains to be understood. While p63 plays a major role in development, p73 plays a major role in tumor suppression as well as in development. Although a functional co-operation is evident between each member of p53 family, additional …

Cellular Function Of The Ia-Motoneuron Circuit Following Peripheral Nerve Regeneration, Katie Leigh Bullinger

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Successful regeneration of a severed peripheral nerve is insufficient to restore the stretch reflex. This deficit occurs despite successful muscle reinnervation and functional restoration of the circuit components. For example, Ia afferents encode muscle stretch, Ia-motoneuron synapses reform to the extent of restoring low frequency transmission, and activation of motoneurons results in muscle force output. However, when recording intracellularly from reinnervated rat motoneurons during ramp hold and release muscle stretch, no excitatory synaptic potentials are detected in 2/3 motoneurons (Haftel et al., 2005), a clear sign that the synapses connecting Ia afferents with motoneurons are physiologically disrupted. This thesis examines …

Ion Transport Mechanisms During Hyposmotic Regulatory And Isosmotic Apoptotic Volume Decreases In A Human Lens Epithelial Cells Line, Ameet Ajit Chimote

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Living cells maintain constant volume in response to physiological stresses by altering trans-membrane ion, solute and water flow. In the present study, early and late membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium (Ki), rubidium (Rbi)-uptake and water content were measured by atomic absorption spectrophotometry and gravimetry, respectively. Intracellular chloride concentration [Cl]i was determined with the fluorescence dye N- (ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), during regulatory volume decrease (RVD) after hyposmotic stress and apoptotic volume decrease (AVD) induced by staurosporine (STP), a protein-kinase inhibitor. Cell water increased in hyposmotic balanced salt solution …

The In Vitro Interactions Between Tubulin And Hiv-1 Rev Require Rev's Multimerization And Arginine-Rich Motifs, Amit Sharma

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Rev is a 13 kDa regulatory HIV protein essential for viral replication. It trans-activates expression of late viral proteins by multimerizing onto target mRNA and promoting their export into the cytoplasm. During attempts to find the solution conditions needed to study Rev structure, Watts et al. (2000) discovered that Rev depolymerizes microtubules (MTs) in vitro through the formation of ringed tubulin intermediates called Rev-tubulin toroids (RTTs). Rev interactions with MTs are specific and are thought to mimic the mechanism of Kinesin-13 proteins, themselves potent MT depolymerases that regulate the assembly of the mitotic spindle. If Rev and Kinesin-13 proteins share …

Small Conductance Calcium-Activated Potassium (Sk) Channels In Mammalian Spinal Motoneurons, Zhihui Deng

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Three homologous small conductance calcium-activated potassium (SK) channel subunits (SK1, SK2, and SK3) are expressed in distinct and overlapping patterns in mammalian central nervous system. SK channels likely mediate the medium afterhyperpolarization (mAHP), which plays an essential role in regulating neuron repetitive firing frequency. In spinal motoneurons (MNs) the mAHP duration is shorter on average in fast (F-type) MNs than that in slow (S-type) MNs. To better understand the molecular basis for mAHP, we determined the expression and sub-cellular distribution of SK channels in normal, axonally-injured, and developing spinal MNs in vivo using immunohistochemistry and quantitative confocal imaging techniques.

SK2 …

Induction Of Socs-1 In Hsv-1-Infected Murine Keratinocytes: A Mechanism Of Inhibition Of Interferon Gamma, Kenneth Gene Frey

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Epidermal cells such as keratinocytes are the major site of Herpes Simplex Virus Type 1 (HSV-1) replication in active primary or recurring herpes infection. In this study, a murine keratinocyte cell line (HEL-30) was shown to be refractory to IFNγ induction of an antiviral state to HSV-1 infection, while IFNγ did induce an antiviral state in a murine fibroblast cell line (L929). Particularly, IFN-gamma and its peptide mimetic protected fibrobasts from viral-induced cytopathic effect while the keratinocytes were destroyed by the infection. Suppressor of cytokine synthesis-1 (SOCS-1) a negative regulator of IFNγ, was hypothesized to be responsible for the refractiveness …

Embryonic And Postnatal Development Of The Neural Circuitry Involved In Motor Control, Valerie Cari Ann Siembab

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The development of locomotion is believed to result from the maturation of the spinal circuits controlling motor output, however little is known about its mechanisms. To shed some light into this process we studied the development of the synaptic connectivity of two spinal inhibitory interneurons. Adult motoneurons are controlled by inhibitory networks that include recurrent and reciprocal inhibition (Pierrot-Deseilligny & Burke, 2005). Each is modulated by different ventral horn spinal interneurons that display synaptic connectivity adapted to their function: Renshaw cells (RCs) mediate recurrent inhibition, receive excitatory inputs from motor axons and inhibit homonymous and synergistic motoneurons; while Ia inhibitory …

Hif-1 Alpha: A Master Regulator Of Trophoblast Differentiation And Placental Development, Kashmira Kulkarni

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Early embryonic development occurs under low oxygen levels. The placenta is an organ transiently formed during pregnancy and plays a crucial role during development of the embryo. Alterations in the placental structure or function have been associated with the pathologies such as preeclampsia in humans. In mammals, the hypoxia inducible factor (HIF) transcription factors have been identified as the major regulators of cellular responses in low oxygen. In the current study, we investigated the effects of low oxygen on the differentiation of trophoblast stem cells, lineage committed trophoblast giant cells and labyrinthine lineage-committed cells. We examined the effects of an …

Central Mechanisms Regulating Pituitary-Adrenal Activity In Infant Guinea Pigs (Cavia Porcellus) During Exposure To Psychological Stressors: Independent And Combined Effects Of Maternal Separation And Novelty, Deborah Suzanne Maken

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Separation from the maternal attachment figure, particularly when it occurs in a novel or threatening environment, reliably increases pituitary-adrenal activity in a number of species, and is thought to increase later susceptibility to psychopathology in humans. However, little is known about the central mechanisms mediating these effects. Therefore, I investigated cortisol and ACTH levels in plasma, corticotropin-releasing factor (CRF) mRNA expression in the hypothalamic paraventricular nucleus (PVN), and c-Fos activity in a circuit [medial amygdala (MeA)- bed nucleus of the stria terminalus (BNST)] thought to drive pituitary-adrenal activity during exposure to psychological stressors, as well as in the PVN. Measurements …

Genetic Differentiation In Ambystomatid Salamanders Across A Fragmented Landscape, Joseph E. Bartoszek

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It is generally recognized that amphibian populations declining across the globe. Habitat loss is the primary cause of decline. The salamander family Ambystomatidae has experienced significantly more rapid decline than the average for amphibians. Coefficients of Conservatism (CoC) have been developed for amphibians in Ohio. These are based primarily on habitat requirements with high CoCs for amphibians with stringent habitat requirements (e.g. vernal pools associated with large, intact, undisturbed forest), and low CoCs for amphibians with less stringent habitat requirements. Genetic diversity in five species and one hybrid complex of ambystomatid salamander in southwest Ohio were examined and compared to …

Characterization Of Excitatory Amino Acid Neurotransmitters At Motoneuron Synapses Contacting Renshaw Cells, Dannette Shanon Richards

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Motoneurons are among the best studied neurons in the central nervous system. The motoneuron synapses have been well characterized in the periphery where they release acetylcholine at the neuromuscular junction. However excitatory amino acids also seem to be released from motoneuron terminals in the periphery, and centrally at their synapses contacting Renshaw cells. Although excitatory amino acids are suggested to be released from motoneuron synapses it is not known which excitatory amino acids (either aspartate or glutamate) are released, nor is the mechanism for their release known. To examine the presence and mechanism of release for aspartate and glutamate at …

The Effects Of Chronic Hypoxia And Substance P On The Chemosensitive Response Of Individual Nucleus Tractus Solitarius (Nts) Neurons From Adult Rats, Nicole L. Nichols

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The chemosensitive responses of individual nucleus tractus solitarius (NTS) neurons from neonatal rats have been extensively studied, but few studies have examined the chemosensitive responses of NTS neurons from adult rats. In addition, environmental conditions have been used to mimic respiratory diseases/disorders in rats to study how the cellular responses of individual neurons change to regulate breathing during pathological conditions. Lastly, it has been shown that substance P release increases in response to hypoxia from peripheral afferents that primarily terminate in the caudal NTS. We studied the effect of chronic hypoxia (CHx) and substance P on the response to hypercapnia …

Novel Redox And Dna-Dependent Conformational Changes In Human Ku, A Dna-Double Strand Break Repair Protein, Jason Alexander Lehman

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Ionizing radiation (IR) and radiomimetic drugs used in cancer chemotherapy cause DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. Ku is a heterodimeric protein comprised of 70 and 80 kDa subunits and recognizes free DNA ends. Once Ku is bound to DNA, it binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and forms a heterotrimeric DNA-PK complex for repair and damage signaling.

We have analyzed the Ku protein using multiple biochemical techniques and uncovered a novel reversible redox change (Andrews, Lehman and Turchi, (2006) JBC 281(19):13596-603). From this data, we hypothesized that a redox-dependent …

Loss Of Hdmx Leads To Alterations In Gene Expression And Inhibition Of Cell Growth In Tumor Cells With Wild-Type P53, Katherine Ann Heminger

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Mutations in the p53 tumor suppressor gene are among the most prevalent molecular abnormalities in human cancer. While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. This dissertation focuses on human tumor cell lines harboring wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. My hypothesis is that loss of HdmX in tumor cells with wild-type p53 and over-expressed HdmX, will activate p53 and induce p53 target genes leading to growth …

Structural And Signaling Elements Important For The Efficient Degradation Of Bhmt Through Macroautophagy, Carol A. Mercer

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Healthy cells maintain a dynamic and responsive intracellular environment that is marked by the synthesis and degradation of proteins, complex macromolecules and organelles. Autophagy, literally "self-eating," is a mechanism that delivers cellular cargo to the lytic compartment for digestion. Defects in the regulation of autophagy have been implicated in pathologies such as cancer and neurodegenerative disease, making the study of its regulation compelling. However, few studies have looked at the regulation of mammalian autophagy as a function of a specific cargo protein. Previous studies had indicated that the metabolic enzyme betaine homocysteine methyltransferase (BHMT) is degraded through an autophagic mechanism. …

Biochemical Characterization Of Htrf1 And Htep1, Two Proteins Involved In Telomere Maintenance, Kambiz Tahmaseb

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Telomeres are the structures that protect the ends of linear chromosomes from fusion and degradation. The telomere consists of tandem repeated DNA sequences that can range from hundreds of bases to kilo-bases depending on the organism. As the cells of an organism replicate their DNA, these repeats are lost due to the end replication problem, where the ends of linear DNA cannot be fully replicated. As the telomeres are shortened through each round of replication, they eventually reach a critical point. Once the telomeres are too short and the cell risks losing coding sequences, a signaling pathway is initiated that …

Investigating The Role Of Subunit Iii In The Structure And Function Of Rhodobacter Sphaeroides Cytochrome C Oxidase, R. Ryan Geyer

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The role of subunit III (SIII) in cytochrome c oxidase structure and function was investigated using enzyme isolated from the bacterium Rhodobacter sphaeroides. Energy minimization calculations suggested that in the absence of SIII, subunit I (SI) adopted a more open conformation. This observation was tested through the use of limited proteolysis using, á-chymotrypsin. The results showed that in the absence of SIII the solution structures of wild-type and I/II oxidase were not significantly different, and that proteolysis occurred exclusively at the N and C-termini of SI. Upon inactivation of I/II oxidase by catalytic turnover, and subsequent digestion with the protease …

Functional Interplay Between Subthreshold Ion Channels In Preautonomic Neurons Of The Hypothalamic Paraventricular Nucleus In Health And Disease Conditions, Patrick M. Sonner

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Under normal conditions, blood pressure is tightly regulated through autonomic tonic and reflex mechanisms. However, when the set-point for blood pressure is chronically elevated, hypertension occurs. Hypertension if untreated can lead to further complications including heart failure, stroke and kidney failure. Elevated sympathetic outflow is known to contribute to the development and/or maintenance of hypertension, and while the hypothalamic paraventricular nucleus (PVN), a preautonomic center, has been implicated in the elevation of sympathetic activity during hypertension, the precise pathophysiological mechanisms underlying sympathoexcitation remain unclear. Subthreshold ion channels, including the A-type K ⁺(I _A) and the T-type Ca ^{2+ …}

Regulation Of Dna Replication Initiation By Histone Acetylation And The Dna Unwinding Element Binding Protein Due-B, Michael George Kemp

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Duplication of the genome during S phase of the mitotic cell cycle begins at thousands of sites along chromosomes termed origins of replication. Although many of the essential protein components catalyzing events at these sites are known and are conserved throughout eukaryotes, the likelihood or efficiency of initiation of DNA synthesis at any given genomic site is expected to be influenced by other novel factors, including aspects of chromatin and DNA structure.

Here I show that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific …

Feasibility Of Radiographic Absorptiometry Of The Mandible As An Osteoporosis Screening Method, Julie A. Skipper

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The purpose of this work was to develop and evaluate single- and dual-energy radiographic absorptiometry (RA) for the measurement of mandibular bone mineral density (BMD), using standard dental equipment, that would allow for inexpensive and widespread osteoporosis screening. Accurately measured x-ray tube spectra are important for validating the simulations used in the design of the method. A constrained least-squares deconvolution technique for reducing the detector-induced blurring of the energy spectrum was developed. Application of this analytic correction to spectra acquired with a NaI-photomultiplier detector improved the measured data by 20-60%. Implementation of the screening method is accomplished by simultaneously acquiring …

Defining Benthic Organism Exposure: Bioavailability And Effects Of Non-Polar Organics, Marc Samuel Greenberg

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Laboratory and field tests were conducted to evaluate the hypothesis that factors such as the total organic carbon (TOC) contents and groundwater-surface water interactions (GSI) in the sediments can affect chemical desorption, bioavailability and benthic organism exposure. Laboratory studies were conducted with the polycyclic aromatic hydrocarbon fluoranthene (FLU) and the herbicide trifluralin (TF). Toxicokinetic parameters were determined for Lumbriculus variegatus and Hyalella azteca in water-only exposures to 0, 5, 20 and 50 µg/L of the compounds and bioaccumulation was measured during exposures to 0, 100 and 200 mg/kg of FLU and TF spiked onto sediments from Lakes Erie and Huron. …