Browsing by Subject "Biomedical Engineering and Bioengineering"
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Item Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB(2001-11-01) Chang, Woo-Jin; Alvarez, Rafael; Mathew, Porunelloor A.; Goldfarb, Ronald H.Chang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.Item Genetic Engineering and the Food Supply(2000-12-01) Kennedy, W. Russ; Samuel F. AtkinsonAs food sources high in the trees became inadequate, our predecessors climbed to the ground and through centuries of adaptation learned to stand upright and cultivate plants. Plant cultivation has been practiced for more than ten thousand years with continuous improvements made to crop plants to meet the growing food needs of human domesticated animal populations. Biotechnology has been practiced for thousands of years with fermentations of fruits and grains to make wine and beer and the use of yeast in baking. More recently, advances in molecular biology allow the analysis and manipulation of genetic material to achieve desired changes in the organism. Transgenics or genetic engineering is the process of identifying specific genetic defects or desirable traits and altering an organism’s DNA by addition or deletion of specific DNA sequences. Nearly 100 million acres (40 million hectares) were planted in transgenic crops in 1999. The largest acreages of more than 40 different transgenic crops grown were in cotton, corn, soybean and rapeseed. Fifty-five percent of all cotton, 50% of soybeans, and 33% of corn grown in the U.S. in 1999 were transgenic varieties. The large plantings stem from fairly straightforward manipulations of single genes, such as the transferring to corn and cotton genetic material from the bacterium Bacillus thuringiensis (Bt) which produces an insecticidal toxin or transferring to the soybean, corn, cotton, sugar beets, and canola a gene with resistance to herbicides, such as glyphosate. The American farmer is perceived to be the beneficiary of lowered production costs primarily through better weed and pest control and a reduction in pesticide use with accompanying environmental improvement. Agro-chemical companies, who for the most part have spearheaded research and development of these crops, became involved because they foresaw a declining market for pesticides. Another area of promise widely discussed in the scientific and popular press is the improvement of food quality and composition resulting from genetic engineering. Because plants and plant products provide much of the world’s food supply, it is only fitting that early applications of this technology be in this area. Recent estimates suggest that the market for transgenic seed has already reached several hundred million dollars per year and that more than 15 million hectares (37 million acres) were grown in the U.S. in 1998. Concerns of food risk to the food supply and environment that using transgenic methods present, although not always science based, have some merit and require careful scientific scrutiny.Item Identification of Oxidized Proteins in Alzheimer's Disease(2002-08-01) Choi, Joungil; Gracy, Robert R.; Harris, B.; Lacko, Andras G.Joungil Choi, Identification of Oxidized Proteins in Alzheimer’s Disease. Doctor of Philosophy (Molecular Biology and Immunology). August, 2002. Pages-110. Tables 8. Figures 24. Oxidative modification of specific proteins is central to the pathology of Alzheimer’s disease (AD). The purpose of this study was to identify the oxidation-sensitive proteins in neuronal cells, fibroblasts from AD subjects, and in the blood of AD patients. In all cases, age-matched non-Alzheimer’s samples were used as controls. Proteomic methods were used to isolate and characterize the oxidized proteins. These included two-dimensional gel electrophoresis, immunolocalization of oxidized proteins and identification by MALDI-TOF mass spectroscopic methods. It was hypothesized that knowledge of these critical oxidation-sensitive proteins would shed light on the underlying mechanism of the disease. In addition, it was postulated that these proteins might prove to be biomarkers for early detection and monitoring the progress of the disease. The results show that two different oxidative stressors (H2O2 generated enzymatically, or the amyloid beta peptide, AB25-35) induce apoptotic cell death and oxidation of specific proteins (heat shock protein 60 and vimentin) in skin fibroblasts from AD subjects and in neuronal cells. In addition, the results indicate that susceptibility of these two proteins to oxidative stress is increased in fibroblasts from AD patients, compared to non-AD controls. Pretreatment with antioxidants (e.g., vitamin E or flavonoids) protect these proteins from oxidative damage. Both heat shock protein 60 and vimentin, have been suggested to function as antiapoptotic proteins. Thus, their oxidative damage could lead to the apoptotic neuronal cell death in Alzheimer’s disease. In the blood plasma of AD subjects, isoforms of fibrinogen gamma chain and alpha-1 antitrypsin were found to be oxidized. These proteins exhibited to a two- to six-fold greater specific oxidation index in plasma from AD subjects when compared to controls. Both of these proteins have been suggested to be implicated in oxidation-mediated damage of inflammation in the AD brain.Item Mechanisms of Photoreceptor Cell Apoptosis(2000-05-01) Crawford, Matthew John; Neeraj Agarwal; Victoria Rudick; Raghu KrishnamoorthyCrawford, Matthew John, Mechanisms of Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May 2000; 168 pp; 3 tables; 23 figures; bibliography, 282 titles. Photoreceptor cell death mediated by programmed cell death pathways is responsible for many disease states of the retina, which result in vision loss. Examples of this include retinal dystrophies and age-related macular degeneration. Correspondingly, the understanding of programmed cell death, or apoptosis, in these cells is important in the formulation of preventative and treatment options. The goals of this dissertation are to characterize a suitable in vitro photoreceptor cell model and explore the molecular mechanisms resulting in apoptotic cell death secondary to oxidative cell death paradigm. Means of interrupting the cell death process were also investigated. An immortalized clonal mouse retinal cell line was shown to express photoreceptor-specific genes and proteins by RT-PCR amplification, Western blot analysis, and immunocytochemical localization. Exposing these cultured cells to visible light resulted in oxidative stress, as exhibited by elevated malonyldialdehyde and reduced gluthathione levels, as well light exposure-dependent apoptosis was shown using multiple techniques which identified fragmentation of chromosomal DNA, a key finding in the apoptotic cell death process. Molecular regulators of apoptotic cell death, including bcl-2 family proto-oncogenes and the nuclear transcription factor NF-kB, were found to be important in oxidative stress-induced pathogenesis of 661 W photoreceptor cells. mRNA and protein levels of the anti-apoptotic proto-oncogene bcl-2 declined following oxidative stress disturbing the balance proto-oncogene regulators and initiating the apoptotic pathway. The nuclear transcription factor NF-kB was found to be constitutionally expressed in the photoreceptor cells with its down-regulation during apoptosis. Permanent transfection of the photoreceptor cells with bcl-2 gene imparted protection from apoptosis and sustained NF-kB levels. The results presented in this dissertation help define the molecular mechanisms which occur during apoptosis of photoreceptor cells. Photo-oxidative stress results in programmed cell death mediated through changes in NF-kB binding activity and bcl-2 family genes. The involvement of caspase-1 in the degradation of NF-kB and the execution of apoptosis is also demonstrated. Over-expression of the proto-oncogene bcl-2 interrupts the apoptotic events, protecting against down-modulation of NF-kb binding activity and cell death. Our proposed mechanism for apoptosis in photoreceptor cells provides several points at which targeted gene expression (bcl-2 or NF-kB), or pharmaceuticals (anti-oxidants, caspase inhibitors, or calcium channel blockers) may prevent apoptotic cell death.Item Molecular Cloning, Expression, and Regulation of the Na+/Myo-Inosiotl Cotransporter Gene(1996-08-01) Zhou, Cheng; Chaitin, Michael; Easom, Richard; Garner, MargaretZhou, Cheng, Molecular Cloning, Expression, and Regulation of the NA+/Myo-Inositol Cotransporter Gene. Doctor of Philosophy (Biomedical Sciences), August 1996. Mammalian cells respond to osmotic stress by accumulation of high concentrations of intracellular osmolytes. Osmotic-induced accumulation of the osmolyte, myo-inositol (MI), is achieved through activation of the NA+/MI cotransporter. Hypertonic stress results in elevated NA+/MI cotransporter mRNA abundance and transcription rate, and increased transporter activity. The goals of this dissertation are to establish the osmoregulation of the NA+/MI cotransporter gene expression in lens cells, and to investigate the transcriptional regulation of the NA+/MI cotransporter gene. Expression of the Na+/MI cotransporter in cultured bovine lens epithelial cells (BLECs) was demonstrated by RT-PCR amplification and Northern blot analysis. Hypertonic stress resulted in induction of the NA+/MI contransporter mRNA abundance in cultured BLECs. The induction patterns of the NA+/MI cotransporter and aldose reductase mRNA abundance by hypertonic stress indicated that osmoregulation of MI and sorbitol accumulations were regulated in concert. Accumulation of MI is an early-onset protective system, which is suppressed by the elevated sorbitol, the late-onset protective system. 5’-RACE analysis indicated that multiple transcription start sites were utilized in controlling of the expression of the NA+/MI cotransporter. Osmotic stress resulted in preferential utilization of a hypertonic promoter a. The bovine NA+/MI cotransporter gene was cloned and analyzed. The regulation of the Na+/MI cotransporter expression was investigated by transient transfection assays using promoter-luciferase constructs. Although multiple promoters were functional in cultured BLECs, only the hypertonic promoter a was osmotically responsive. Characterization of this osmotic-responsive element(s) between -536 to -300 bp upstream of the hypertonic transcription start site a. The studies presented in this dissertation refined the osmoregulation of the Na+/MI cotransporter gene expression. Hypertonicity induces MI accumulation by activation of an osmotic-responsive promoter. The consequences of the activation of this promoter lead to more cotransporter mRNA, more cotransporter protein, and higher transporter activity, resulting in accumulation of a higher concentration of intracellular Mi.Item Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model(2002-12-01) Kern, Jami RaDel; Thomas Yorio; Robert W. Gracy; Peter B. RavenKern, Jami RaDel, Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model. Doctor of Philosophy (Biomedical Sciences, Biochemistry and Molecular Biology), December 2002, 156 pp., 1 table, 27 illustrations, references, 112 titles. The focus of these studies was to characterize a novel connective tissue model for use in experiments examining possible contraction initiators in the wound healing process, i.e. endothelin-1 (ET-1). Through these studies, it has been shown that use of a telomerized dermal fibroblast cell line addresses the concerns relating to variations due to heterogeneity of normal human cells cultured in vitro, without creating a cancerous cell line or interfering with normal phenotypic changes. In addition, the incorporation of telomerized cells into our TE, which does not spontaneously contract (US Patent #6471958), provides a unique model to study the contraction and scar formation process. Using the TE populated with hTERT fibroblasts, an innovative technique was developed to identify the initiation of tissue contraction using an optical fiber interferometry system. The process allows observation of contraction within five minutes of stimulus addition and also enables continuous data capture over a period of several hours. The greatest strength of this system is its sensitivity, since optic interferometer allows measurement of displacement (contraction) to the tens of nanometers. Along those lines, the current studies have identified ET-1 as a potential early initiator in wound healing and suggest a novel pathway through which it functions. This proposed mechanism includes both direct effects of ET-1 through the Rho-associated kinase pathway and indirect effects potentiated by TGF-β. Future studies addressing whether TGF-β converges on the Rho-associated kinase pathway or acts independently through other signaling mechanisms should be initiated. The discovery of early initiators of tissue contraction is essential in the identification of potential therapeutic targets in the quest to reduce prolonged and severe tissue contracture and scaring.Item Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing with the Potential for Automation(2002-05-01) Marshall, Pamela L.; Eisenberg, Arthur; Martin, Michael W.; Wordinger, Robert J.Marshall, Pamela. Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing With the Potential for Automation. Master of Science (Forensic Genetics). May, 2002. In 1998, within the United States, it is estimated that a rape occurred every 2.3 minutes. In 1995, according to the Bureau of Justice Statistics, an estimated 350,000 rapes or sexual assaults (R/SA0 were experienced by persons age 12 or older. Of the estimated 100,000 R/SA reported, there were only approximately, 25,000 cases analyzed by crime labs nationwide. The majority of crime laboratories throughout the U.S., especially those in major metropolitan cities, have a significant backlog of unresolved R/SA cases. With the implementation of the Convicted Offender Database (CODIS), it is essential that all R/SA cases by analyzed, especially those lacking a known suspect. The comparison of the short tandem repeat (STR) profiles derived from sperm DNA recovered from evidentiary material with CODIS samples would provide the police with critical investigative leads resulting in the identification of the assailant. The goal of this research is to develop a cellular sorting method for the isolation of sperm cells from sexual assault samples which will: 1) take advantage of differentiating features (extracellular antigenic sites) for complete separation of cell types, 2) provide a more efficient means of sperm recovery, increasing DNA yield from the male fraction, and 3) ensure the DNA isolation process is compatible with the amplification of the CODIS core STR loci. Overall, the proposed technique will increase the probability of success in the analysis of sexual assault case samples. (NIJGrant #: 2000-IJ-CX-K009).Item Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B(2003-12-01) Confer, Nils Forgard; Alvarez, Rafael; Ben S. Aar…; Wu, MingehiConfer, Nils Forgard, Protein-Protein Interactions Between Poly(ADP-ribose) Polymerase-1 and DNA Polymerase B. Doctor of Philosophy (Biomedical Sciences), December 2003, 114 Pages, 22 Figures, 1 Graph, and 80 References. The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase B (pol B) function. Activity gels were used to measure the DNA polymerase activity of pol B following protein-(ADP-ribosyl)ation. However, the fraction of pol B molecules (ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose) Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol B to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol B, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol B activity. Moreover, I also observed the specific molecular association of PARP-1 is specifically proteolyzed into peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either capase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol B as supported by co-immunoprecipitation/immune-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol B that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degredation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol B, and inhibits DNA repair associated pol B activity to irreversibly switch the fate of cell from BER toward chromatin degradation and, eventually, programmed cell death.Item The Urokinase Plasminogen Activator System in NK Cells: Its Role in Invasion And Its Regulation by IL-2(2002-05-01) Al-Atrash, Gheath; Goldfarb, Ronald H.; Kitson, Richard P.; Mazar, Andrew P.Al-Atrash, Gheath, The Urokinase Plasminogen Activator System in NK Cells: Its Role in Invasion and its Regulation by IL-2. Doctor of Philosophy (Biomedical Sciences), May 2002; 202 pp.; 2 tables; 20 figures; bibliography, 143 titles. Adoptively transferred natural killer (NK) cells can infiltrate tumors and directly kill malignantly transformed cells without prior sensitization. This makes NK cells ideal for cancer immunotherapy, not only for their tumoricidal capacities, but also as drug carriers. This dissertation investigates the role of NK cell urokinase plasminogen activator (uPA) system in NK cell invasion through extracellular matrices (ECMs), its cooperation with matrix metalloproteinases (MMPs), the mechanism by which interleukin-2 (IL-2) upregulates NK cell uPA/uPAR, and the in vivo antitumor therapeutic potential of NK cells as drug delivery vehicles. uPA and its receptor uPAR were detected in human and rat NK cells using RT-PCR, casein plasminogen zymography, western blots, and fluorescence microscopy. In vitro invasion assays showed a role for the uPA system in NK cell invasion, alone and in cooperation with MMPs: this was achieved by using selective plasmin inhibitors in combination with selective MMP inhibitors in ECM/Matrigel invasion assays. uPA’s regulation by the ECM proteins collagen type IV, laminin, and fibronectin was investigated and results show a downregulation of NK cell uPA mRNA by these proteins. IL-2, however, a potent NK cell stimulator, increases both uPA and uPAR, coinciding with an increase in NK cell invasion. This IL-2 upregulation has transcriptional and posttranscriptional components, the latter mediated by uPA and uPAR destabilizing mRNA binding proteins. The use of NK cells as drug delivery vehicles was illustrated by in vivo studies which demonstrated that NK cells linked to doxorubicin were more efficacious in prolonging the survival of tumor bearing mice than either treatment alone. The work presented in this dissertation has substantial impact on the field of adoptive immunotherapy for cancer treatment. Engineering NK cells to transiently express high amounts of uPA and/or uPAR may increase the invasive capacities of NK cells, resulting in greater infiltration of tumors by ex-vivo activated NK cells. Moreover, this enhanced infiltration may allow a greater delivery of anticancer drugs to established metastatic tumors. This can potentially lead to more efficacious and possibly curative NK mediated adoptive immunotherapy, thereby constituting a novel means to overcome current limitations to NK cell-mediated adoptive therapy of cancer metastases.