Browsing by Subject "Molecular Genetics"
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Item A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression(1995-08-01) Zeng, Hong; Stephen R. Grant; Walter McConathy; Richard EasomZeng, Hong, A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression. Master of Science (Biomedical Science), August, 1995, 85 pp., 2 tables, 20 illustrations, bibliography, 90 titles. A cultured myocardial cell model was used to examine a potential role of calcium-dependent protein kinases and phosphatases in regulating the induction of the atrial natriuretic factor (ANF) gene mediated through adrenoreceptor signaling. In primary culture, rat neonate cardiomyocytes supplemented with phenylephrine (PE) following transfection (24 h) with a full length ANF promoter-reporter construct, showed elevated levels of promoter activity when compared to transfected cardiomyocytes cultured in the absence of PE. Prazosin, a dedicated α1-antagonist, completely blocked the transcriptional induction mediated through PE stimulation. Two different calcium mobilizing agents, BAY K8644 and gramicidin D, significantly reduced PE-stimulated ANF promoter activity. The over-expression of co-transfected exogenous CaM kinase II isoforms resulted in transcriptional silencing of PE-induced promoter activity for cardiac ANF. Transfection of a constitutively active, mutant form of the calcium-dependent phosphatase 2B, calcineurin, gene also transcriptionally silenced ANF gene expression. Exposure of PE-induced cardiomyocytes to either FK-506-treated cells in the absence of PE exposure suggesting that transcriptional silencing may be mediated through a transcriptional repression mechanism. Taken together, these results suggest that the activation of a Ca2+-dependent nuclear signaling pathway mediated through either CaM kinase II or calcineurin leads to complete transcriptional silencing of the embryonic ANF gene expression.Item A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli(2002-12-01) Weilbacher, Thomas; Jerry SimeckaWeilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.Item Aging of the Thymic Epithelial Progenitor Pool is Determined by the p63-FoxN1 Regulatory Axis(2015-05-01) Burnley, Preston I.; Dong-Ming SuThe immune system is composed of various effector cells and molecules that must work in concert in order to protect the body against infections, auto-reaction, and tumor occurrence. These responses can be divided into two categories – innate and adaptive immunity. The innate response is the host’s first line of defense towards a pathogen by providing a physical and chemical barrier against infection. Once activated, innate cells such as macrophages and dendritic cells can engulf the bacterium, degrade it, and secrete proteins to destroy the pathogen. Although this response occurs immediately after an encounter with a pathogen, the innate immunity is neither long-lasting nor specific. In contrast, the adaptive immune response is initiated when the innate immune response is unsuccessful in eliminating the infection, allowing for recognition and response tailored for a particular pathogen. The cells that make up the adaptive response all originate from a common lymphoid progenitor found in the bone marrow. From this precursor arise natural killer (NK) cells (part of the innate response) and T and B lymphocytes. The T lymphocytes originate from the bone marrow but undergo development in the thymus, hence the name T cells. B lymphocytes, on the other hand, originate and develop in the bone marrow. With the exception of NK cells, these adaptive immune cells require antigen presentation in order to become activated. Once the T and B cells have matured and become activated they can work together to clear the infection by secreting cytokines and antibodies. The most important aspect of the adaptive immune response is its ability to produce immunological memory. Memory T and B cells are able to ensure a rapid and effective response to a second encounter, providing long-last immunity. Unfortunately, this well-ordered process, specifically the development of T cells, becomes compromised during aging. This is due to the fact that thymic involution (or shrinking of the thymus) occurs at the onset of puberty and continues throughout the lifespan, which is primarily resulted from age-related defect in thymic epithelial cells (TECs). The thymus is crucial for the generation of T cells so any compromise to the organ results in changes in the T cells, which can possibly lead to immune insufficiency and autoimmunity [1]. Additionally, these conditions are exacerbated with age [2, 3]. This research project will focus on the molecular mechanism(s) responsible for thymic involution. To do so, we focused on TECS and two genes associated with the homeostatic maintenance of the thymic microenvironment, p63 and FoxN1. These genes regulate the proliferation and differentiation, respectively, of thymic epithelial cells (TECs), thereby maintaining a properly functioning thymus. For this study we will utilize our mouse model (FoxN1 conditional knockout, FC) extensively because it mimics an aged thymus. This model allows us to study the thymic microenvironment of a mouse with a defect in the FoxN1 gene.Item Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur EisenbergThe AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.Item Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of NonHemolytic, Catalase-Deficient Variants of Staphylococcus aureus(1999-06-01) Crum, Russell M.Crum, Russell M., Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of Nonhemolytic, Catalase-Deficient Variants of Staphylococcus aureus. Masters of Science (Microbiology). June 1999. Pages-101. Tables-15. Figures-10. A Tn917 transposon mutant of Staphylococcus aureus S6C was isolated and analyzed due to its deficiency in hemolysin and lipase activities. The transposon insertion did not occur in any of the known genetic regulators, which suggested the insertion occurred in a novel regulator of at least, hemolysin and lipase activities. One end of the region where the insertion occurred was isolated, sequenced, and compared with known DNA databases. Sequence comparisons revealed the insertion occurred in one of six rRNA DNA operons, which was confirmed by Southern analysis. Transduction of the transposon insertion back into the parental strain did not result in a mutant phenotype thereby indicating that the transposon insertion into a rRNA DNA operon was not responsible for the observed mutant phenotype. Further analysis of the parent strain, S. aureus S6C, revealed a population of four relatively stable variants differing in their hemolysin and catalase activities. These data suggest that the Tn917 mutant was one of these four S6C variants.Item Approaches to Cloning and Identification of the Ligand for Natural Cytotoxicity Receptor NKp44(2008-07-01) Horton, Nathan C.; Harlan Jones; Stanley Stevens; Raghu KrishnamoorthyHorton, Nathan C., Approaches to Cloning and Identification of the Ligand for the Natural Cytotoxicity Receptor, NKp44. Masters of Science (Microbiology & Immunology), July 2008, 64 pp., 22 illustrations, 37 titles. Natural Killer (NK) cells represent a specialized lymphoid population that mediate innate immune responses against tumor or virally infected cells. NK cell cytotoxicity is regulated by inhibitory and activating receptors. Activating receptors include the Natural Cytotoxicity Receptors (NCRs), 2B4, and NKG2D. The NCRs play a key role in recognition and killing of tumor cells and include the receptors NKp30, NKp46, and NKp44. The ligands for the NCRs are not yet known. NKp44 is of particular interest because it is only expressed on activated NK cells, and is implicated in increased cytotoxicity and HIV infection. To identify and clone the ligand for NKp44, a recombinant fusion protein containing the extracellular domain of NKp44 was constructed and used to identify a cell line, DB, expressing a ligand for NKp44. A directional complimentary DNA (cDNA) library was constructed from this cell line and screened by mammalian expression cloning, resulting in the isolation of several putative cDNA clones of NKp44 ligands.Item Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism(2002-05-01) Campbell, Rowan Stewart; Arthur J. Eisenberg; Bruce Budowle; John PlanzCampbell, Rowan Stewart, Automatable Virtual Array Screening System For Rapid Analysis of Mitochondrial DNA Polymorphism. Doctor of Philosophy (Biomedical Sciences), May, 2002, 156 pp., 11 tables, 48 illustrations, bibliography, 96 titles. The goal of this research project was to develop alternative methods to traditional forensic mtDNA sequence analysis. Conventional forensic mtDNA analysis requires the direct sequencing of Hypervariable Region I and Hypervariable Region II in both the forward and reverse directions. This method is time consuming, labor intensive and expensive. Two methods for determining mtDNA haplotypes through the direct interrogation of Single Nucleotide Polymorphisms with HVI and HVII have been developed. A Sequence Specific Oligonucleotide Hybridization assay was developed on the Luminex 100™ flow cytometer, as well as a Single Base Extension assay developed for the ABI Prism® 310 Genetic Analyzer. The SNP typing of mtDNA sequences can provide a significant benefit in many forensic and human identification cases. The reassociation of mass disaster remains, mass grave analysis, and the screening of large numbers of crime scene samples are examples of their potential application. Their inclusion as a standard screening tool would be high beneficial since more extensive DNA analysis would be reserved for those samples that possess the greatest evidentiary value. In a blind study of 50 samples, the Sequence Specific Oligonucleotide Hybridization assay incorrectly identified the mtDNA haplotypes in 7 samples, whereas the Single Base Extension assay correctly identified each of the SNP positions interrogated. The SNaPshot™ primer extension assay was approximately 20-25 times more sensitive than the standard sequencing approach. This would suggest that this system could be a viable alternative to sequence analysis when samples are limited, as well as being more robust in detection and typing of heteroplasmic sites. A statistical evaluation of the SNP panels revealed that the genetic diversity estimated for the 50 Southwestern Hispanic samples tested was 0.9624 for the primer extension array and 0.9559 for the hybridization-based array. The probability of two randomly selected individuals from a population group having the same mtDNA haplotype was 0.0568 for the Single Base Extension assay and 0.0632 for the Sequence Specific Oligonucleotide Hybridization assay. A forensic mtDNA SNP array consisting of the positions evaluated in this study could provide a reasonable alternative to the full sequencing of the HVI and HVII regions.Item Changes in Mammalian Chromatin Structure as a Function of Protein-Poly(ADP-Ribosyl)ation by Endonuclease Digestion(2004-06-01) Perez-Lamigueiro, Maria A.; Alvarez, Rafael; Das, Hriday K.; Basu, AlakanandaPerez-Lamiguerio, Maria A., Changes in Mammalian Chromatin Structure as a Function of Protein-poly(ADP-ribosyl)ation by Endonuclease Digestion. Master of Science (Biochemistry and Molecular Biology), June 2004. 66 pages, 12 illustrations, Bibliography, 45 titles. Mammalian chromatin was exposed to either Deoxyribonuclease I or Micrococcal Nuclease digestion as a function of time of incubation and enzyme concentration. Endonuclease enzymatic reactions were stopped with EDTA. Samples were run in 1.5% agarose gels and the oligonucleosomal electrophoretic migration patterns compared. Endonuclease experiments were carried out with rat liver chromatin pre-incubated in the presence or absence of 200 μM βNAD+. A solution of 1.0 mM benzamide was used to stop enzymatic modification. The electrophoretic observations demonstrated a faster and increased degradation of chromatin when proteins were poly(ADP-ribosyl)ated prior to digestion. These results support the hypothesis that that the covalent poly(ADP-ribosyl)ation of chromatin proteins, particularly histones, induces a more relaxed structure, rendering chromatin more sensitive to endonuclease digestion.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Differentiation of Vascular Smooth Muscle Cells(2004-05-01) Deaton, Rebecca Ann; Dillon, Glenn; Shepard, Allan; Mallet, Robert T.Rebecca Ann Deaton, Characterization of the role of PKN in TGF-beta 1-mediated differentiation of vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), May 2004, 178 pp, 5 tables, 34 illustrations, references, 197 titles. Differentiated vascular smooth cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors. SMCs retain the ability to de-differentiate into an “immature” proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanism involved in maintain SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-B1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-B1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs. These characteristics are indicative of the differentiated phenotype. The effects of TGF-B1 can be blocked by pretreatment of the cells with either HA1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-B1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM a-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF-GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-B1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK ½. Co-expression of dominant negative p38 MAPK is sufficient to abolish PNK-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-B1 activates RhoA and PKN to promote differentiation of SMCs.Item Comparison of Next Generation Sequencing Methodology on the Ion PGM™ System Performance versus that on the Sanger Sequencing Method for HV1 and HV2 Regions of mtDNA(2015-05-01) Argueta, Wendy C.; Arthur J. Eisenberg; Michael Allen; Raghu R. KrishnamoorthyAnalysis of mitochondrial DNA in forensic applications has enabled the identification of a missing person through comparison with additional maternal relatives. Most forensic applications are based on sequencing of both hypervariable regions of the mtDNA. Sequencing of these regions has been commonly done using Sanger-type sequencing (STS) methodology, which is expensive, time-consuming and laborious. Next Generation Sequencing (NGS) technology, such as the Ion Torrent PGM™ System platform, overcomes most of these issues. In this study, samples from the Guatemalan population (n=40) were sequenced with both Ion Torrent PGM™ technology and STS methods. A high level of consistency (98%) was observed among data derived from both methods. Most of the discrepancies were point heteroplasmy, which were more easily detected by PGM™ technology. In terms of performance, the NGS method was shown to be quick, with high-throughput and more efficient compared to the traditional STS method. More accurate and reliable sequencing data were obtained from the Ion Torrent PGM™ method due to its high level of coverage. Sequencing data for all individuals, representing 19 different family groups, were obtained using the NGS technology. Sequence polymorphisms were detected in 55 positions, from which 26 were observed only in relatives belonging to the same family and were not observed for any other family group. In a forensic context, haplotype specific polymorphisms are the basis for identification and comparison between evidence and reference samples purposes. Haplotypes between maternally related individuals were consistent in 18 family groups.Item Construction of a Cost Effective Nested-PCR Reaction for Use with the Applied Biosystems AmpFLSTR Identifiler Kit(2005-12-01) Mikeska, Margo M.; John Planz; Joseph Warren; Arthur EisenbergHuman STR analysis has greatly increased the ability to perform identity testing for many different situations. These situations include, but are not limited to, the identification of individuals involved in violent crimes, establishing paternity, and identification of unknown human remains. The most common type of DNA information currently used for identity testing is the short tandem repeat, or STR. STR testing utilizes the number of repeating units in the DNA to assign an allele. Alleles from several different loci are used to establish a genetic profile. Currently, the United States used a standard of 13 different DNA loci to establish identity. These 13 loci can be typed by using a number of different multiplex kits such as the Applied Biosystems Profiler Plus, Cofiler, and Identifiler Kits [1,2]. The 13 loci were selected based on a number of parameters. Each locus was required to be polymorphyic, and a tetranucleotide repeat. The loci also could not display any linkage between each other and extensive population studies had to be conducted to both verify the absence of linkage and to establish allelic frequencies [1]. The goal of this research was the construction of a more cost effective method of utilizing the Applied Biosystems Identifiler Kit. Across the country there is a large backlog of samples that need to be processed in order to obtain a genetic profile. If these samples could be tested using a more cost effective method, more funding could be directed to other endeavors. Paternity testing, as well as some research endeavors could be conducted at a fraction of the cost, leaving resources for other projects or additional staff. Although it would be inadvisable to use this technique on forensic samples, the implications on paternity and research samples would be positive. This research attempted to design a nested PCR reaction and subsequently dilute the Applied Biosystems Primers in order to reduce the cost. The first step was to design new primers for the first round of PCR, followed by testing of those primers. The new primers then required optimization so that they all worked effectively together. After optimization was accomplished, the Identifiler primers were diluted until loci began dropping out of the genetic profile.Item Discovery and Characterization of Tetranucleotide Short Tandem Repeats in North American Bears (Ursids)(2015-05-01) Graham, Michelle L.; John V. PlanzAccurate individual identification is essential to wildlife crime investigations and conservation genetics. Current methodology utilizes dinucleotide short tandem repeats (STRs) that can be difficult to type accurately and have high mutation rates; however, tetranucleotide STRs have greater stability and allele diversity. The main objective of this study was to identify potential tetranucleotide STR loci and internal variants for the American black bear, brown bear, and polar bear. Barcoded genome libraries were prepared for each species from extracted and enzymatically fragmented DNA, size selected, quantified, enriched using biotinylated RNA baits to capture twelve common mammalian sequence motifs, and massively parallel sequenced. One potential locus was identified using the NextGENe® software and six potential loci were identified using algorithm mining.Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.Item Evaluation of a Novel Multiplex Ministr System for Analysis of Degraded and Low Copy DNA Samples(2006-05-01) Orcutt, Joseph L.; Arthur Eisenberg; Joseph Warren; John PlanzOrcutt, Joseph L., Evaluation of a Novel Multiplex MiniSTR System for Analysis of Degraded and Low Copy DNA Samples. Masters of Science (Forensic Genetics), May, 2006, 78 pp., 25 tables, 14 figures, bibliography, 20 titles. The goal was to evaluate the performance of a novel miniSTR multiplex system for the analysis of degraded and low quantity DNA samples. Three studies were designed to evaluate this new miniSTR kit: 1. A concordance study to insure that the profiles generated are identical to those with currently used STR kits; 2. A dilution study to identify the sensitivity limits of the multiplex system, and 3. The ability to generate profiles from DNA isolated from skeletal remains which had previously given incomplete profiles using conventional STR kits. The results indicate that the Applied Biosystems new miniSTR multiplex system will provide a valuable tool for forensic scientists to obtain genetic data from challenging casework samples.Item Evaluation or the Gold-Plated Silver Sample Block on the GeneAmp PCR System 9700 Thermal Cycler(2003-05-01) Moore, Melody Ann; Arthur Eisenberg; Joseph Warren; John PlanzMoore, Melody Ann. Evaluation of the Gold-plated Silver Sample Block on the GeneAmp PCR System 9700 Thermal Cycler. Master of Science (Forensic Genetics), May 2003, 49 pages, 9 figures, 3 tables, 2 appendices, 24 references. The GeneAmp PRC System 9700 Thermal Cycler has been introduced with interchangeable silver and gold-plated silver sample blocks. To validate the new gold-plated silver sample block on the System 9700, amplifications were performed on both the gold-plated silver and the previously validated silver sample blocks. PCR amplifications using the AmpFLSTR Profiler Plus ID COfiler, Identifiler and SGM Plus typing kits (Applied Biosystems, Forster City, CA) were performed. Electrophoretic characteristics such as allele concordance, peak heights, and peak height ratios were used to discern any differences in the amplification capabilities of the two sample blocks. The results demonstrate that the PCR reactions on both silver and gold-plated silver blocks are equivalent. In addition, the data obtained (allele cells, peak heights, peak height ratios) from both Macintosh and Windows NT platforms were identical for each amplification kit, indicating concordance between the software packages of each operating system. The results of this study demonstrate the interchangeability of the silver and gold-plated sample blocks.Item Genetic Diversity of Easter Island (Rapanui) Population from Identifiler® Plus autosomal, Y-filer®, and Y-Plex™ 6 Y-STR Loci(2015-05-01) Guadian, Laura; Chakraborty, Ranajit; Budowle, Bruce; Hodge, Lisa M.This study investigated the genetic diversity of the Easter Island (Rapanui) population using data on 15 autosomal Short Tandem Repeats (STRs) typed with the commercial STR kits Identifiler® Plus and 23 Y-chromosome STRs typed using Y-filer (17 loci) and Y- PLEX™ 6 (6 loci). The analysis was conducted using genotype and haplotype data of 122 presumably unrelated individuals that included 48 males and 74 females. This study: (i) examined if Easter Island population had reduced genetic diversity in comparison with cosmopolitan populations such as Mainland Chilean, Polynesian, European, and African; (ii) compared genetic affinity of the Easter Island population with historically related cosmopolitan populations; and (iii) investigated the forensic utility of autosomal STRs and Y-STRs in the Easter Island population.Item Identification of Novel Genes Involved in Escherichia coli Biofilm Formation(2003-05-01) DesPlas, Rebecca L.; Jerry Simecka; Ming-Chi WuDesPlas, Rebecca L., Identification of Novel Genes Involved in Escherichia coli Biofilm Formation. Master of Science (Microbiology), May 2003, 77 pp., 6 tables, 11 figures, references, 55 titles. Transposon mutagenesis using a miniTn10::camR transposon generated 800 random insertion mutants displaying altered biofilm phenotypes as compared to the parent strain, TRMG F/M. Transduction of the resistance marker confirmed approximately 150 biofilm mutants. Amplifications of the insertion sites, nucleotide sequencing and BLAST searches against E. coli K-12 genomic databases, identified118 of these sites. Many of the interrupted genes are not known to be associated with biofilm formation. Four mutations were transduced into E. coli K-12 MG1655, creating altered biofilm phenotypes. A plasmid clone of the nhaAR operon complemented the corresponding mutations. Results indicate that the genes identified in this study influence biofilm formation. However, further studies are needed to determine the degree of impact in a wild type strain background.Item Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, ZygmuntAdkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.Item MIEN1 Drives Breast Cancer Invasion by Regulating Cytoskeletal-Focal Adhesions Dynamics(2015-05-01) Kpetemey, Marilyne F.; Vishwanatha, Jamboor K.; Clark, Abbot F.; Basu, AlakanandaIn the recent years, Migration and Invasion Enhancer 1(MIEN1) has emerged as a potential biomarker and a plausible target in breast cancer. Located in the 17q12-21 region of the human chromosome, next to the Her-2/neu loci, MIEN1 presents a robust expression in breast carcinomas; however is completely absent or low in the normal tissues. MIEN1 is post-translationally modified by geranyl-geranyl transferase-I (GgtaseI), which adds isoprenyl group to the carboxyl-terminal of the protein. Prenylated MIEN1 then associates with the inner leaflet of the plasma membrane and acts as an adaptor protein triggering downstream signaling through the Akt/NF-kB axis to regulate the expression of key proteases and angiogenic factors like MMP-9, uPA and VEGF. In migrating cells, MIEN1 enhances filopodium formation at the leading edge. Aside from its prenylation and redox-active motifs, MIEN1 also contains a canonical ITAM, reported to be associated with epithelial-to-mesenchymal transition. Although the role MIEN1 in cell migration and invasion is well known, the underlying molecular mechanisms remain elusive. Here, we show that MIEN1 interacts with Annexin A2, a cytoskeletal protein and a regulator of the plasminogen/plasmin system in breast cancer cells to increase migration and invasion. We confirmed that MIEN1 regulates actin dynamics by associating with cytoskeletal effectors in the lamellum. We also show that MIEN1 expression redirects breast tumor cell migration toward a collective migration. Our studies validate MIEN1-ITAM and CAAX as key motifs to MIEN1-induced functions. In conclusion, our findings confirm the role of MIEN1 in the remodeling of the actin cytoskeleton during motility. Furthermore it attests to previous findings suggesting that motility patterns depend on various environmental factors along with regulatory genes involved. Our study demonstrates an interesting example from cell biology where adaptor proteins regulate various signaling pathways and control cellular processes through protein-protein interactions.Item Molecular Characterization of a Second Superoxide Dismutase Gene (sodM) of Staphylococcus Aureus and Effects of SodM and SodA on Oxidative Stress Resistance and Virulence(2001-11-01) Valderas, Michele Wright; Porunelloor A. Mathew; Richard Easom; Jerry SimeckaValderas, Michelle Wright., Molecular Characterization of a Second Superoxide Dismutase Gene(sodM) of Staphylococcus aureus and Effects of SodM and SodA on Oxidative Stress Resistance and Virulence. Doctor of Philosophy (Biomedical Sciences), November, 2001, 191 pp., 4 tables, 20 illustrations, bibliography, 389 titles. A second gene for superoxide dismutase (SOD) in S. aureus (sodM) was cloned and characterized. This gene was found to be unique to S. aureus among the staphylococci. S. aureus is the first gram-positive bacterium reported to contain two or more SOD activities. The three native SOD enzymes observed for S. aureus can be accounted for by two distinct genes, sodM and sodA. The SodM and SodA proteins form homodimers, but their subunits also interact to form an active heterodimeric SOD. The deduced amino acid sequence from each gene and the relative insensitivity to hydrogen peroxide and potassium cyanide indicated that the S. aureus SODs utilize manganese as a metal ion cofactor. Additionally, viabilities of the soda and sod double mutants, but not the sodM mutant were drastically reduced under conditions of oxidative stress in early exponential growth. However, only the double mutant was affected when oxidative stress was applied in the late-exponential and stationary phases of growth. It was determined, therefore, that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under conditions of oxidative stress, becomes a major source of SOD activity during the late-exponential and stationary phases of growth such that the viability of the S. aureus sodA mutant is maintained. Experiments examining the roles of sodM and sodA in virulence determined the ability of S. aureus to cause disease in the mouse lung and subcutaneous abscess formation in mice was unaffected by sod mutation. Lack of SOD activity, however, results in enhanced clearance of S. aureus within the lung and also promotes killing of the organism by mouse macrophage cell lines and human polymorphonuclear leukocytes.