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Characterizing The Dynamics And Functional Role Of Site-Specific Phosphorylation Of G Protein-Coupled Receptors, John M. Busillo

Full-Text Theses & Dissertations

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) and the subsequent recruitment of arrestin is a well-established paradigm that initiates the process known as desensitization. However, an emerging theme in GPCR regulation is the possibility of differential regulation dictated by the phosphorylation pattern elicited by the different members of the GRK family. Therefore, we have used small interfering RNA-mediated knock down of the GRKs and arrestins in an attempt to better understand how phosphorylation regulates the activity and signaling of the M3 muscarinic acetylcholine receptor (M3 mAChR) and CXCR4, two receptors endogenously expressed in HEK293 cells.

Using a …

Urine Protein Electrophoresis: Comparison Of Agarose Gel And Capillary Zone Electrophoretic Techniques., Anne-Marie Curtin

Theses

Proteinuria is the excess excretion of protein in the urine and is found in a variety of conditions including renal diseases and neoplastic conditions such as multiple myeloma (MM). The investigation of proteinuria type through the use of electrophoresis is a useful and widely used non-invasive diagnostic tool. The aim of this study was to compare various agarose gel and capillary zone electrophoresis techniques in order to establish a method choice for the separation of protein in routine laboratories. Comparisons were made based on urine monoclonal peak quantification and protein fractions detected. An examination of a capillary zone electrophoresis (CZE) …

Development Of An Automated Method For Csf Bilirubin Measurement., Helen Caverley

Theses

Subarachnoid haemorrhage (SAH) is a potentially fatal condition which can be difficult to accurately diagnose. Patients often present with a second bleed which can be more damaging than the initial bleed. Diagnosis of SAH centres on computed tomography (CT) scans. However, 2% of patients positive for SAH will present as negative on CT scans. It is recommended that these patients have lumbar puncture and the CSF is analysed for bilirubin. The presence of bilirubin is suggestive of SAH. Spectrophotometric scanning involves expensive equipment which is often not available in routine settings. A need exists for a robust, inexpensive, automated method …

Design Of A Multiplex Pcr Assay For The Detection Of Neisseria Gonorrhoeae., Elizabeth J. O'Callaghan

Theses

The incidence of N. gonorrhoeae infections in Ireland has been steadily increasing since 2003. Culture is currently the preferred method of diagnosis, however the sensitivity of culture can be low. In recent years there has been increased interest in non-culture techniques such as polymerase chain reaction (PCR). Many of the PCR assays available have been shown to cross-react with commensal organisms, producing false positive results. This study aimed to improve the detection of N. gonorrhoeae by designing a multiplex PCR assay that used two N. gonorrhoeae genes as targets, thereby providing dual detection and confirmation of a positive result. PCR …

Cold Atmospheric Pressure Air Plasma Jet For Medical Applications, Juergen Friedrich Kolb, A.-A H. Mohamed, R. O. Price, R. J. Swanson, A. Bowman, R. L. Chiavarini, Michael W. Stacey

Bioelectrics Publications

By flowing atmospheric pressure air through a direct current powered microhollow cathode discharge, we were able to generate a 2 cm long plasma jet. With increasing flow rate, the flow becomes turbulent and temperatures of the jet are reduced to values close to room temperature. Utilizing the jet, yeast grown on agar can be eradicated with a treatment of only a few seconds. Conversely, animal studies show no skin damage even with exposures ten times longer than needed for pathogen extermination. This cold plasma jet provides an effective mode of treatment for yeast infections of the skin.

Development Of Srap, Srap-Rga, Rapd, And Scar Markers Linked With A Fusarium Wilt Resistance Gene In Eggplant, Nedim Mutlu, Filiz Hatice Boyacı, Münevver Göçmen, Kazım Abak

Department of Biochemistry: Faculty Publications

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melon­gena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F2 and BC1 populations of eggplant segregating for …

Improved Constrained Global Optimization For Estimating Molecular Structure From Atomic Distances, Terri Marie Grant

Mathematics & Statistics Theses & Dissertations

Determination of molecular structure is commonly posed as a nonlinear optimization problem. The objective functions rely on a vast amount of structural data. As a result, the objective functions are most often nonconvex, nonsmooth, and possess many local minima. Furthermore, introduction of additional structural data into the objective function creates barriers in finding the global minimum, causes additional computational issues associated with evaluating the function, and makes physical constraint enforcement intractable. To combat the computational problems associated with standard nonlinear optimization formulations, Williams et al. (2001) proposed an atom-based optimization, referred to as GNOMAD, which complements a simple interatomic distance …

Radiocarbon Evidence For The Importance Of Surface Vegetation On Fermentation And Methanogenesis In Contrasting Types Of Boreal Peatlands, J. P. Chanton, P. H. Glaser, L. S. Chasar, David J. Burdige, M. E. Hines, D. I. Seigel, L. B. Tremblay, W. T. Cooper

OES Faculty Publications

We found a consistent distribution pattern for radiocarbon in dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and methane replicated across spatial and temporal scales in northern peatlands from Minnesota to Alaska. The ¹⁴C content of DOC is relatively modern throughout the peat column, to depths of 3 m. In sedge-dominated peatlands, the ¹⁴C contents of the products of respiration, CH₄ and DIC, are essentially the same and are similar to that of DOC. In Sphagnum- and woody plant-dominated peatlands with few sedges, however, the respiration products are similar but intermediate between the ¹⁴C contents of …

Naturally Present Fatty Acids As Internal Calibrants For Fourier Transform Mass Spectra Of Dissolved Organic Matter, Rachel L. Sleighter, Georgina A. Mckee, Zhanfei Liu, Patrick G. Hatcher

Chemistry & Biochemistry Faculty Publications

The analysis of dissolved organic matter ( DOM) by Fourier transform ion cyclotron resonance mass spectrometry ( FTICR- MS) has gained wide interest recently, driven primarily by its ultrahigh resolving power and mass accuracy. Accurate calibration of mass spectra is a key step to successfully decipher the DOM components. We propose a simple and accurate method to internally calibrate the peaks in the complex spectra without the need to add a calibrant. Mass spectra of DOM samples from the Dismal Swamp, Virginia, and the lower Chesapeake Bay display the presence of naturally occurring fatty acids which can be readily recognized …

Functional Domains Of The Fatty Acid Transport Proteins: Studies Using Protein Chimeras, Concetta C. Dirusso, Dina Darwis, Thomas Obermeyer, Paul N. Black

Department of Biochemistry: Faculty Publications

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of …

The Human Hyaluronan Receptor For Endocytosis (Hare/Stabilin-2) Is A Systemic Clearance Receptor For Heparin, Ed Harris, Janet A. Weigel, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K_d for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and …

7Β-Hydroxy­Artemisinin, Paulo B. Carvalho, Bo Liu, Yunshan Wu, John S. Williamson, Mitchell A. Avery

Office for Research Publications and Presentations

Crystals of the title compound [systematic name: (3R,6R,7S,8aR,9R,12aR)-7-hydr­oxy-3,6,9-trimethyl­octa­hydro-3,12-ep­oxy[1,2]dioxepino[4,3-i]isochromen-10(3H)-one], C₁₅H₂₂O₆, were obtained from microbial transformation of artemisinin by a culture of Cunninghamella elegans. The stereochemistry of the compound is consistent with the spectroscopic findings in previously published works. A weak O—H⋯O hydrogen bond occurs in the crystal structure, together with intermolecular C—H⋯O hydrogen bonds.

Molecular Characterization Of Trypanosoma Cruzi And Shed Vesicle Components Involved In Host Immunomodulation And Cell Invasion, Ernesto Satoshi Nakayasu

Open Access Theses & Dissertations

Chagas disease caused by Trypanosoma cruzi is a devastating infectious disease with millions of cases in Latin America, and recently became a public health concern in United States and Europe. Although many efforts have been made for the development of an effective immunotherapy, currently there is no human vaccine for Chagas disease. Thus, the treatment is based only on two drugs that have limited efficacy and in some cases present severe side effects. One restriction for the rational approach to develop new therapies against this disease is the limited information about the proteins, glycolipids and protein posttranslational modifications expressed by …

Immuno-Opioid Interaction A Potential Connection, Linda Staikos-Byrne

Seton Hall University Dissertations and Theses (ETDs)

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M3-Muscarinic Receptor Activation Of Erk And Cell Growth Requires Calcium/Calmodulin-Dependent Protein Kinases In Mcf-7 Cells, John M. Schmitt, Ellen Abell, Andrea Wagner

Faculty Publications - Department of Biological & Molecular Science

Abstract The extracellular signal-regulated protein kinase (ERK) signaling pathway is found in diverse cells throughout the human body. ERK activation has been implicated in breast cancer cell growth and proliferation. Studies have shown that ERK is activated by carbachol, a G Protein-Coupled Receptor (GPCR) agonist, which increases intracellular calcium in MCF-7 cells. The calcium/calmodulin-dependent protein kinase (CaM K) family of proteins including CaM KK, CaM KI, and CaM KII can be activated by increased intracellular calcium. Our goal was to determine whether CaM Ks may be responsible for ERK activation and cell proliferation in carbachol-treated MCF-7 cells and evaluate which …

Regulated Expression Of Adenoviral Vectors-Based Gene Therapies: Therapeutic Expression Of Toxins And Immune-Modulators, James Curtin, Marianela Candolfi, Mariana Puntel, Weidong Xiong, Akm Ghulam Muhammad, Kurt Kroeger, Sonali Mondkar, Chunyan Liu, Niyati Bondale, Pedro Lowenstein, Maria Castro

Articles

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the "OFF" state, and expression should quickly reach therapeutic levels in the "ON" state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first …

Gender Bias Of Cardiovascular Risk In Caucasian Type Ii Diabetics: A Haemostatic Explanation?, William Quirke

Theses

People with type II diabetes are disproportionately affected by cardiovascular disease compared to those without diabetes. The relative risk of cardiovascular disease caused by type II diabetes is higher in females than in males. This risk has not been fully explained by traditional risk factors and attention has turned to the possible role of haemostatic abnormalities in explaining the excess cardiovascular morbidity and mortality among diabetic females. We examined specific parameters of the coagulation (factor VII, factor VIII, von Willebrand factor, fibrinogen) and fibrinolytic (plasminogen activator inhibitor- 1, thrombin-activatable fibrinolysis inhibitor) system in 113 Irish Caucasian type II diabetics to …

Identification Of Lactic Acid Bacteria In Michigan Cherry Wines, Emily Henk, Margaret Dietrich, Terri Weese

Student Summer Scholars Manuscripts

Many factors contribute to the final flavor of wine. One factor is malolactic fermentation, during which lactic acid bacteria (LAB) transform the harsh tasting malic acid into a more drinkable lactic acid in grape wine. The role of LAB in the production of cherry wine is completely unknown. The goal of this study is to identify the species of LAB in cherry wine and compare them to those found in grape wines. Bacteria from cherry wine were grown on general media plates and plates fortified with malic acid, which may provide optimal growing conditions for the LAB. To identify the …

Chiral Recognition Of Amino Acids By Use Of A Fluorescent Resorcinarene, Gerald I. Richard, Hadi M. Marwani, Shan Jiang, Sayo O. Fakayode, Mark Lowry, Robert M. Strongin, Isiah M. Warner

Chemistry Faculty Publications and Presentations

The spectroscopic properties of a chiral boronic acid based resorcinarene macrocycle employed for chiral analysis were investigated. Specifically, the emission and excitation characteristics of tetraarylboronate resorcinarene macrocycle (TBRM) and its quantum yield were evaluated. The chiral selector TBRM was investigated as a chiral reagent for the enantiomeric discrimination of amino acids using steady-state fluorescence spectroscopy. Chiral recognition of amino acids in the presence of the macrocycle was based on diastereomeric complexes. Results demonstrated that TBRM had better chiral discrimination ability for lysine as compared to the other amino acids. Partial least squares regression modeling (PLS-1) of spectral data for macrocycle-lysine …

Modulation Of Fast-Inactivating Potassium Channels By Accessory Subunits And Fatty Acids : A Role For Calcium?, Michelle M. Drzewiecki

Honors Theses

Potassium channel interacting proteins (KChIPs) co-assemble with Kv4 a subunits to form native complexes that encode considerable components of neuronal somatodendritic A- type K+current (Is,J in neurons, and transient outward current (ho) in cardiac myocytes. The binding of KChIPs to the cytoplasmic N-termini of Kv4 a subunits enhances surface expression by facilitating intracellular trafficking, stimulates subunit assembly, and regulates the functional gating properties of Kv4 channels. KChIPs, like many other neuronal calcium sensor proteins, contain four EF-hand calcium binding sites; two of which (EF-3 and 4) have particularly high affinity for calcium. Because Ca2+ is an important signaling molecule for …

Transcriptional Regulation Of Lamb3 By P53, Meghna Jani

Browse all Theses and Dissertations

The p53 tumor-suppressor plays a very important role in the prevention of cancer and it is known that about 50% of all human tumors possess p53 mutations. Although mutations in p53 are most prevalent in human cancers, inactivation of wild-type p53 occurs through many different mechanisms that are independent of p53 mutation or deletion. In an effort to determine novel p53 target genes, our lab employed a microarray method in which p53 was re-activated by RNAi mediated knockdown of Hdm2 and HdmX in MCF7 human breast cancer cell line, harboring wild-type p53 and elevated levels of Hdm2 and HdmX. Gene …

Ancient Dna Identification Of Early 20th Century Simian T-Cell Leukemia Virus Type 1, Sebastien Calvignac, Jean-Michel Terme, Shannon M. Hensley, Pierre Jalinot, Alex D. Greenwood, Catherine Hanni

Biological Sciences Faculty Publications

The molecular identification of proviruses from ancient tissues (and particularly from bones) remains a contentious issue. It can be expected that the copy number of proviruses will be low, which magnifies the risk of contamination with retroviruses from exogenous sources. To assess the feasibility of paleoretrovirological studies, we attempted to identify proviruses from early 20th century bones of museum specimens while following a strict ancient DNA methodology. Simian T-cell leukemia virus type 1 sequences were successfully obtained and authenticated from a Chlorocebus pygerythrus specimen. This represents the first clear evidence that it will be possible to use museum specimens to …

Validation Of A Custom-Made Microarray To Study Human Intestinal Microflora, Harshavardhan Kenche

Browse all Theses and Dissertations

Intestinal microflora refers to all the different species of bacteria that reside in the human gut and is an important organ of the human body because almost all the digestive reactions of the host occur in the intestine. The bacteria of the intestine play a key role in this process by supplementing the intestine with various enzymes and proteins that are required for the digestive process. At the same time, these bacteria were shown to be implicated in a variety of gastrointestinal disorders like Irritable Bowel Syndrome, Inflammatory Bowel Disorder and Gastrointestinal Cancer, but with the current knowledge about the …

Studying Potential Drug Interactions Involved In The Regulation Of The Diaphanous-Related Formins, Brittany E. Benson, Bradley J. Wallar

Student Summer Scholars Manuscripts

Diaphanous-related formins (DRFs) are a conserved family of proteins that are involved in the regulation of cellular shape, motility, and cell division by regulating the structure of the cellular “skeleton” (cytoskeleton); any disruption of this regulation can result in cell death. Normally, the DRFs are kept in an inactive state by the intramolecular binding of two regions of the protein: the Diaphanous Autoregulatory Domain (DAD) and Diaphanous Inhibitory Domain (DID). This binding can be alleviated by various naturally-occurring mechanisms and proper regulation of the activity of these proteins is vital to cell survival because prolonged activation of DRFs can result …

The Role Of Protein Disulfide Isomerase (Pdi) In Oxidative Folding, Veronica Gonzalez

Open Access Theses & Dissertations

Protein Disulfide Isomerase is a highly evolved oxidoreductase enzyme that has the capability of oxidizing, reducing, and isomerizing disulfides in substrate. PDI is organized into several domains denoted a, b, b', a' and c. These domains are believed to have different functions but all must be present for full PDI activity. In this manuscript we recorded the ability of PDI b' to catalyze oxidative folding in fully reduced RNase A. We also examined competition between thiol-disulfide shuffling and conformational folding by PDI. This competition creates a rough effect of a highly efficient enzyme in two different substrates: RNase A and …

Giardia Lamblia: Genomic And Molecular Analyses Of Flippase, Diana Leticia Villazana-Kretzer

Open Access Theses & Dissertations

Giardia lamblia, is an intestinal protozoan parasite responsible as a major cause of water-borne intestinal infection in humans (giardiasis). It has been proposed earlier that Giardia has limited lipid synThesis ability and therefore relies on preformed lipid molecules from outside sources. It has also been proposed that lipids are taken up through passive and active transport, and giardial flippasse play a significant role in this process. This study describes the identification and characterization of a putative homologue of flippase gene from Giardia. Flippase is an enzyme that functions as a trans-bilayer transport system facilitating the flip flop diffusion of phospholipids …

Mdm2 Amplification In Nih3t3l1 Preadipocytes Leads To Mdm2 Elevation In Terminal Adipogenesis, Vaughn Litteral

Browse all Theses and Dissertations

The p53 protein is a tumor suppressor protein that is mutated or non-functional in nearly all cancers. The Mdm2 protein has the ability to functionally inactivate p53 and these two proteins have been studied extensively in the context of cellular proliferation. In this study, expression of the murine double minute 2 (mdm2) gene was examined in the mouse NIH3T3L1 cell line. Under the proper conditions, the immortalized NIH3T3L1 cells have the ability to differentiate from fibroblasts to adipocytes (Green et al., 1975). This well characterized cell line provides an excellent model to study mdm2 in differentiation. While evaluating the regulation …

In Search For New P53 Regulated Genes, Meldrick Daniel Mpagi

Browse all Theses and Dissertations

The p53 tumor suppressor protein has the ability to transactivate its target genes whose gene products are involved in carrying out cell cycle arrest, apoptosis, DNA repair, and senescence. Here, I report that two genes may be p53 regulated. Utilizing a microarray method to search for novel p53 target genes, I was able to identify a possible transcriptional target of p53 being solute carrier family 1a1 (SLC1a1). Along with that finding I also identified an E2F-target gene, minichromosome maintenance 10 (MCM10), as being p53 regulated. Gene expression profiling of MCF7 breast cancer cells treated with RNAi targeting Hdm2 and HdmX …

Involvement Of Inflammation In Tau Pathology And Mechanisms That Drive Neurodegeneration In Alzheimer's Disease, Lisette T. Arnaud

Dissertations, Theses, and Capstone Projects

Two of the hallmarks of neurodegenerative disorders, such as Alzheimer's disease (AD), are (1) the appearance of proteinaceous deposits in inclusion bodies containing aggregates of ubiquitinated proteins and (2) activated microglia and astrocytes surrounding the diseased neurons. In Alzheimer's disease, the intracellular inclusion bodies are known as neurofibrilary tangles (NFT). The mechanisms leading to inclusion body formation and their role in the progression of neurodegeneration are still largely unknown.

Many of the proteins that accumulate in inclusion bodies depend on the ubiquitin/proteasome pathway (UPP) for their degradation. This pathway is responsible for the bulk (∼80%) of intracellular protein degradation. Because …

Comparative Genomic Analyses Of Copper Transporters And Cuproproteomes Reveal Evolutionary Dynamics Of Copper Utilization And Its Link To Oxygen, Perry Ridge, Yan Zhang, Vadim N. Gladyshev

Vadim Gladyshev Publications

Copper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. …