Molecular Biology Commons^™

Isolation And Functional Mapping Of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Dna-Damage Complexes, Sarah Saionz Durkin

Theses and Dissertations in Biomedical Sciences

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral trans-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events.

We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian …

The Use Of Proteomic Technologies To Identify Serum Glycoproteins For The Early Detection Of Liver And Prostate Cancers, Elizabeth Ellen Schwegler

Theses and Dissertations in Biomedical Sciences

The application of proteomic technologies to identify serum glycoproteins is an emerging technique to identify new biomarkers indicative of disease severity. Many of these newly evolving protein-profiling methodologies have evolved from previous global protein expression profiling studies such as those involving SELDI-TOF-MS technologies. Though the SELDI approach could distinguish disease from normal by utilizing protein patterns as shown herein with the HCC study of chapter II, it was unable to offer sequence information on the selected peaks, and did not have the ability to analyze the entire dynamic range of the serum/plasma proteome. To address these deficiencies, new strategies that …

Never Let Me Clone? Countering An Ethical Argument Against The Reproductive Cloning Of Humans, Yvette Pearson

Philosophy Faculty Publications

In the March 2006 issue of EMBO reports, Christof Tannert, a bioethicist at the Max Delbrück Research Centre in Berlin, Germany, presented a moral argument against human reproductive cloning on the basis of Immanuel Kant’s categorical imperative (Tannert, 2006). In this article, I address some problems with Tannert’s views and show that our concerns about this prospective procedure should prompt us to scrutinize carefully the conventional procreative practices and attitudes. Indeed, if we set aside objections that are grounded in genetic determinism, many of the offensive features of human cloning are identical to problems with procreation by more conventional means, …

2-Aminoethoxydiphenyl Borate As A Prototype Drug For A Group Of Structurally Related Calcium Channel Blockers In Human Platelets, Yuliya Dobrydneva, Christopher J. Abelt, Beth Dovel, Celina M. Thadigiri, Roy L. Williams, Peter F. Blackmore

Chemistry & Biochemistry Faculty Publications

We have synthesized a series of 2-aminoethoxydiphenyl borate (2-APB, 2,2-diphenyl-1,3,2-oxazaborolidine) analogs and tested their ability to inhibit thrombin-induced Ca²⁺ influx in human platelets. The analogs were either synthesized by adding various substituents to the oxazaborolidine ring (methyl, dimethyl, tert-butyl, phenyl, methyl phenyl, and pyridyl) or increasing the size of the oxazaborolidine ring to seven- and nine-membered rings. NMR analysis of the boron-containing analogs suggests that each of them exist as a ring structure through the formation of an N -> B coordinate bond (except for the hexyl analog). The possibility that these boron-containing compounds formed dimers was also …

Chromosomal Localization Of The Islet Neogenesis Associated Protein (Ingap) Gene In Syrian Hamster By Tyramide Signal Amplification-Fluorescence In Situ Hybridization (Tsa-Fish), Sallie A. Smith

Biological Sciences Theses & Dissertations

Diabetes mellitus is a group of conditions characterized by hyperglycemia due to an inability to produce or properly utilize insulin. The majority of cases fall into two categories, Type I and Type 2. Type I results from the autoimmune destruction of pancreatic β-cells of the islets. The beta cells are the exclusive source of insulin and the patient becomes entirely dependent on exogenous insulin to survive. Patients with Type 2 are distinguished by insulin resistance, a condition that develops due to the inability of the body to effectively use the insulin being produced. The β-cells gradually lose their ability to …

Defensin In The Ticks Dermacentor Variabilis And Ixodes Scapularis, Shane Michael Ceraul

Theses and Dissertations in Biomedical Sciences

This dissertation presents the identification, molecular structure and expression of defensin, an antimicrobial peptide, in the ticks, Dermacentor variabilis and Ixodes scapularis. A 624 bp cDNA molecule for defensin was cloned and sequenced from hemocytes, fat body and midgut of unstimulated D. variabilis ticks. Defensin peptide was detected only in the hemocyte lysates. These results suggest that the defensin from D. variabilis is synthesized and stored in the hemocytes, although the role of other tissues is not excluded.

The ability of the juvenile stage ticks to express the defensin gene was confirmed. Detection of the defensin transcript in all …

The Antitumor Agent, Arglabin-Dma, Preferentially Induces Apoptosis In Human Colon Tumor Cells, Sung Wook Kwon

Theses and Dissertations in Biomedical Sciences

Arglabin-DMA, an analog of farnesyl pyrophosphate (FPP), reportedly inhibits farnesyltransferase (FTase) directly by competitively blocking the binding of Ras protein and its posttranslational modification, as suggested in previous studies. But, the mechanisms by which Arglabin-DMA inhibits tumor growth in vivo and in vitro are still relatively poorly characterized. To determine the mechanism by which this drug inhibits tumor growth, the effects of Arglabin-DMA in two human colon tumor cell lines (mutant K-ras HCT 116 and wild-type ras HT-29) were explored on cell proliferation, apoptosis, and cell cycle kinetics in vitro. In cell viability studies, we showed that Arglabin-DMA …

Mechanisms Of Cell Death Initiated In Herpes Simplex Virus Thymidine Kinase Expressing Colon Tumor Cells Treated With Ganciclovir And Ucn-01, Christina Elizabeth Ahn

Theses and Dissertations in Biomedical Sciences

Metastatic colon carcinoma is the second leading cause of death from malignancy in the United States, and development of more effective treatments is essential. Heterologous expression of Herpes Simplex Virus Thymidine Kinase (HSVtk) in combination with the prodrug, ganciclovir (GCV), has shown great promise for the genetic therapy of many cancers, but most patients have had only a partial or minimal response to the therapy. After screening a panel of two drug combinations, our laboratory has shown that the combination of GCV and the protein kinase inhibitor UCN-01 (7-hydroxystaurosporine) enhances tumor cell death more effectively than either drug alone. However …

Regulation Of Sparc Gene Expression By The Activator Protein 1 Transcription Factor, Joseph William Briggs

Theses and Dissertations in Biomedical Sciences

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotypic changes related to malignant progression including a shift to estrogen independent growth, increased cell motility and invasion. Concurrent with these phenotypic changes are alterations to cellular gene expression patterns. One gene that becomes highly upregulated is SPARC (secreted protein acidic and rich in cysteine). Increased SPARC expression is associated with malignant progression in a variety of different cancers, although little is known regarding the mechanisms of SPARC gene regulation. Therefore, the objectives of this study were: (1) to determine the mechanisms by which c-Jun …

Tumor Response Tcf-4/Β-Catenin Regulatory Elements For Enhancing Cancer Gene Therapies, Saurabh Kumar Gupta

Theses and Dissertations in Biomedical Sciences

Mutations in the adenomatous polyposis coli gene are frequently associated with progression of colon carcinoma and most other types of epithelial carcinomas. This usually results in stabilization of β-catenin protein levels, followed by transactivation of Tcf-4/β-catenin responsive genes. The effectiveness of a Tcf-4/β-catenin transcriptional enhancer element in combination with a c-fos or carcinoembryonic antigen promoter was tested for its ability to act as a tumor specific regulator of gene expression in a panel of human tumor and normal cell lines. Luciferase reporter assays indicated enhanced activity of the Tcf-4/β-catenin transcriptional element only in tumor cell lines, with minimal activities in …

On Thacker's Biomedia (2004), Nicholas Ruiz Iii

Reconstruction: Studies in Contemporary Culture

[First paragraph]

Only we could have done it. The Code (DNA and its analogs, etc.) that we perhaps only narrate, never forgetting all of the fictions that narration portends, today consciously edits the text that evolution has edited unconsciously for eons. Thacker considers how humans -- perhaps little more than interactive Code, manipulate the Code -- with the technology of the Code. No other faction of Code has yet to make such heady ‘gains.’ Where posthumanity was always a literary endeavor, a literary bypass, out of instrumental science and into fictive states, it now is a technical endeavor, encompassing a …

Paradigm Shifts And New Worldviews: Kuhn, Laudan And Discovery In Molecular Biology, Priya Venkatesan

Reconstruction: Studies in Contemporary Culture

[Editors' Introduction]

The author poses an alternate set of ideas concerning paradigms, normal science, research traditions and scientific revolution on the basis of the unique and inherently distinctive development of molecular biology and genetics; for, a new set of conceptual tools concerning scientific progress are required for an adequate understanding of how biological processes were elucidated under the scientific auspices of the central dogma of the genetic code. This essay concentrates on a limited study of three aspects in genetics: the study of chromosome structure, dynamics and function, reverse transcriptases, and the discovery of RNA catalysts. In attempting to analyze …

Stimulation Of Capacitative Calcium Entry In Hl-60 Cells By Nanosecond Pulsed Electric Fields, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (similar to100 nM), while the uptake of …

Computational Protein Biomarker Prediction: A Case Study For Prostate Cancer, Michael Wagner, Dayanand N. Naik, Alex Pothen, Srinivas Kasukurti, Raghu Ram Devineni, Bao-Ling Adam, O. John Semmes, George L. Wright Jr.

Mathematics & Statistics Faculty Publications

Background: Recent technological advances in mass spectrometry pose challenges in computational mathematics and statistics to process the mass spectral data into predictive models with clinical and biological significance. We discuss several classification-based approaches to finding protein biomarker candidates using protein profiles obtained via mass spectrometry, and we assess their statistical significance. Our overall goal is to implicate peaks that have a high likelihood of being biologically linked to a given disease state, and thus to narrow the search for biomarker candidates.

Results: Thorough cross-validation studies and randomization tests are performed on a prostate cancer dataset with over 300 patients, obtained …

Dissecting The Loci Of Low-Level Quinine Resistance In Malaria Parasites, Michael T. Ferdig, Roland A. Cooper, Jianbing Mu, Bingbing Deng, Deirdre A. Joy, Xin-Zhuan Su, Thomas E. Wellems

Biological Sciences Faculty Publications

Quinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of …

Hybridization Between The Watersnakes Nerodia Sipedon And Nerodia Fasciata, In The Carolinas: A Morphological And Molecular Approach, Konrad Mebert

Biological Sciences Theses & Dissertations

A few traditionally applied diagnostic characters of color pattern were compared with an additional set of morphological and genetic characters to evaluate differences between Nerodia sipedon and N. fasciata and to study the dynamics across their hybrid zone in the Carolinas. Many of the morphological characters exhibited significant interspecific differences, although only the number of dorsally complete crossbands (CBa) was diagnostic by itself. A discriminant function analysis of morphological characters was successful in separating both taxa. Species-specific nuclear markers, identified by the AFLP (Amplified Fragment Length Polymorphism) technique were nearly fixed and served as diagnostic markers. They revealed extensive introgression …

Hyaluronate Lyase Gene Of Streptococcus Pyogenes: Molecular Characterization And Regulation, Sheryl Lynne Walton

Theses and Dissertations in Biomedical Sciences

Streptococcus pyogenes causes an assortment of diseases ranging from pharyngitis to necrotizing fasciitis. This bacterium has the ability to elaborate several extracellular products capable of causing tissue damage; one of which is a hyaluronate lyase. Little information is available regarding the regulation of streptococcal hyaluronidase. As such, the expression of hylA in the hyaluronate lyase-producing strain ATCC 10403 and in strains that do not produce detectable enzyme was analyzed by RT-PCR and quantitative PCR. Hyaluronate lyase transcript was detected throughout growth for all strains; however, expression of hylA in the enzymatically inactive strains, 71698 and SF370, was determined …

The Quantitative Assessment Of Osteoinductivity Of Human Demineralized Bone Matrix And Cdna Array Analysis Of Osteogenic Differentiation In Human Periosteal Cells, Sittisak Honsawek

Theses and Dissertations in Biomedical Sciences

Demineralized bone matrix (DBM) has been extensively utilized in orthopeadic, periodontal, and maxillofacial applications. DBM comprises bone morphogenetic proteins (BMPs) that are essential regulators for endochondral bone formation. The current study was undertaken to establish the quantitative in vitro assays for determining the osteoinductive potential of human DBM and the use of cDNA gene expression array technology for examining gene expression profiles during osteoblast differentiation in human periosteal cells.

BMP-4 is one of the most osteoinductive factors and has been investigated for clinical applications. For this reason, BMP-4 was designated as the osteoinductive protein for use in the quantitative in …

Protocols For Disease Classification From Mass Spectrometry Data, Michael Wagner, Dayanand Naik, Alex Pothen

Mathematics & Statistics Faculty Publications

We report our results in classifying protein matrix-assisted laser desorption/ionizationtime of flight mass spectra obtained from serum samples into diseased and healthy groups. We discuss in detail five of the steps in preprocessing the mass spectral data for biomarker discovery, as well as our criterion for choosing a small set of peaks for classifying the samples. Cross-validation studies with four selected proteins yielded misclassification rates in the 10-15% range for all the classification methods. Three of these proteins or protein fragments are down-regulated and one up-regulated in lung cancer, the disease under consideration in this data set. When cross-validation studies …

Multiple Transporters Associated With Malaria Parasite Responses To Chloroquine And Quinine, Jianbing Mu, Michael T. Ferdig, Xiaorong Feng, Deirdre A. Joy, Junhui Duan, Tetsuya Furuya, G. Subramanian, L. Aravind, Roland A. Cooper, John C. Wootton, Momia Xiong, Xin-Zhuan Su

Biological Sciences Faculty Publications

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CO), quinine (ON) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CO resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CO and ON. Such molecules may contribute to increasing incidences of ON treatment failure, the molecular basis …

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

Involvement Of Platelet-Derived Growth Factor, Pdgf Receptor Signaling, Focal Adhesion Kinase And Src In Pressure-Induced Vascular Smooth Muscle Hypertrophy, Darian Clark Rice

Theses and Dissertations in Biomedical Sciences

Elevated blood pressure is associated with varying degrees of arterial remodeling. The mechanisms by which extracellular mechanical stress is converted into intracellular alterations in signal transduction and gene expression have yet to be fully elucidated. Our goal was to investigate the early events in the vascular smooth muscle response to acute hypertension and to identify mediators involved in long-term hypertensive remodeling.

In the acute phase of hypertension we targeted cell surface integrin and growth factor receptors thought to be mechanically sensitive. The signaling molecules FAK, Src and ERK-MAPK are known to be triggered by integrin engagement and growth factor receptor …

Prostate Specific Membrane Antigen (Psma): Immunoassay Development And Characterization Of Transcriptional Regulation, Zhen Xiao

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCA) is the most common cancer and the second leading cause of death among American men. The high mortality is greatly attributed to the lack of early detection tools and effective treatment for metastasis and relapses. Biomarkers that can discriminate benign from malignant tumor and signal the development of androgen independent and metastatic tumor are needed. A biomarker designated prostate specific membrane antigen (PSMA) has the potential to fulfill this need. The objective of this study is to develop a clinically useful immunoassay for quantitation of serum PSMA and to study the molecular mechanism underlying the upregulation of …

Fluorescence In Situ Hybridization On Sperm Using Alkaline Denaturation, L. J. Aridgides, M. Stacey, L. Brihn, D. Scott, C. Osgood

Bioelectrics Publications

Fluorescence in situ hybridization (FISH) permits the detection of unique DNA sequences of target DNA within single cells. FISH allows the identifica­tion of DNA sequences within individ­ual cells and readily identifies genetic changes within a mixed population of cells. However, problems arise in main­taining nuclear morphology, particular­ly following harsh denaturation proce­dures where cells are heated to 80°C, causing the nuclear boundary to be­come blurred or the cell to completely burst. In an attempt to preserve cellular morphology and high hybridization ef­ficiency, particularly in fresh tissue samples, we evaluated a FISH protocol using alkaline denaturation in place of heat.

Map Kinases In Cynomolgus Monkey Sperm Hyperactivation, Emily Nicole Haynes

Biological Sciences Theses & Dissertations

Mammalian sperm exhibit characteristic motility patterns, termed hyperactivated (HA) motility, associated with completion of capacitation. In cynomolgus monkey (Macaca fascicularis) sperm, this HA motility is dependent in vitro upon the addition of exogenous cyclic nucleotide mediators, caffeine and dibutyryl-cyclic adenosine monophosphate (dbcAMP). Previous reports have shown protein tyrosine phosphorylation to be an integral component of this caffeine- and cAMP-stimulated HA motility. This study investigated the involvement of the mitogen-activated protein (MAP) kinase-signaling cascade. Semen specimens were collected in Talp-HEPES medium from proven breeders via electroejaculation. After washing, sperm were incubated in the presence and absence of the MAP …

Regulation Of The Hcmv Ul98 Promoter By Cooperation Of The Cyclic Amp Response Element And Gamma Interferon Response Element, Tabmitha Patricia Young Jervey

Theses and Dissertations in Biomedical Sciences

The HCMV UL98 early alkaline exonuclease gene promoter was examined to determine the DNA sequences as well as viral and/or cellular proteins functional in the regulation of this early gene. To assess promoter activation, UL98 promoter sequences were first cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with expression plasmids which express the HCMV major immediate early (IE) proteins 1E72 and 1E86. To more specifically determine the importance of individual cis-acting elements in UL98 promoter activation, the promoter region underwent mutagenesis to delete or alter sequences. The variant promoters were again cloned into a reporter-CAT construct and …

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Interactions Among Murine Cytomegalovirus Us22 Family Gene Products That Influence Viral Pathogenesis, Zaruhi Karabekian

Theses and Dissertations in Biomedical Sciences

Cytomegalovirus (CMV) is a complex, ubiquitous herpesvirus that is characterized by acute, chronic, and latent infections. Monocytes-macrophages are the key target cell type involved in pathogenesis, which is most effectively studied using the murine model of CMV infection. Previously three murine CMV (MCMV) genes (M139, M140, and M141) were identified to regulate viral expression in cultured macrophages and in mice. These genes are members of the US22 gene family with respect to HCMV homology. There is no function assigned to the proteins encoded by these genes. However, deletion of M139, M140, and M141 significantly curtails growth of MCMV in macrophages …

Detection Of Aneuploidy For Chromosomes 7 And 8 Using Fluorescence In Situ Hybridization In Patients With Aplastic Anemia And Sequencing Of The Mitotic Checkpoint Gene Hbub1, Laura Jane Aridgides

Theses and Dissertations in Biomedical Sciences

Aplastic anemia (AA) is characterized by complete bone marrow failure. Progression to myelodysplastic syndromes (MDS) and acute nonlymphocytic leukemia (ANLL) occurs frequently. At the time of transformation, cytogenetic abnormalities are common. Detection of cytogenetic abnormalities prior to leukemic transformation may indicate future disease progression. Karyotype analysis is the current method of choice to evaluate chromosome aberrations. However, fluorescence in situ hybridization (FISH) is more sensitive in detecting these abnormalities.

hBUB1, a mitotic spindle checkpoint gene, was shown to be mutated in two colorectal cancer cell lines with high levels of aneuploidy (Cahill, et al., 1998). Although theoretically possible, conclusive …

Characterization Of The Human Cytomegalovirus Ul75 Late Gene Promoter, Bernard J. P. Mcwatters

Theses and Dissertations in Biomedical Sciences

Gene expression during productive infection by the human cytomegalovirus (HCMV) occurs in an ordered and sequential manner, beginning with immediate early (IE), then early (E) and finally late (L) gene expression. Significant work has addressed the regulation of IE and E gene expression while relatively little work has addressed the control of late gene expression. In order to further address HCMV late gene expression, the promoter of the HCMV UL75 (glycoprotein H, gH) late gene was characterized. The data obtained in this study were combined with observations made in two other studies that have addressed HCMV late gene expression to …