Browsing by Subject "Genetics and Genomics"
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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 DNA-Based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains(2007-12-01) Ambers, Angie; Joseph Warren; John Planz; Arthur EisenbergAmbers, Angie, A DNA-based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains. Master of Science (Forensic Genetics), December, 2007, 63 pages, 13 tables, 19 figures, references, 38 titles. In mass death scenarios, human remains are often fragmented, scattered, and commingled. Ascertaining the number of victims and determining the victims’ identities in such scenarios is a challenging task. A DNA-based screening tool used early in the investigation of mass disasters or mass graves would provide a relatively quick way to initially assess casualty numbers and separate remains for further analysis. Such a tool would promote the most efficient allocation of resources and speed the identification process. The multiplex designed here incorporates a few genetic loci that show high variability in the human population, giving it sufficient discriminatory power for separation of commingled remains. Specifically, the multiplex includes the amelogenin sex-determining locus, D3S1358, and a 3’ (CA)n dinucleotide repeat in the mitochondrial D-loop. Further optimization/validation studies need to be conducted, and a fourth locus (D5S818) may need to be considered to increase the tool’s power of discrimination.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 A Semi-Automated Methodology for Extraction of DNA from Human Skeletal Remains(2013-05-01) Mize, Mary L.; Arthur EisenbergThe current methodology used by the University of North Texas Center for Human Identification Missing Persons Laboratory (UNTCHI) to recover DNA from skeletal remains is time-consuming, laborious and not readily amenable to automation. The constraints of the current process limit the number of samples that can be analyzed. The results of this study show that extractions performed with the AutoMate Express™ Forensic DNA Extraction System (Life Technologies, Carlsbad, CA) can produce comparable DNA quantity and quality to the current procedure used by UNTCHI. The utilization of the AutoMate Express™ Forensic DNA Extraction System in our operational laboratories would help streamline the process of DNA extraction from human skeletal remains and potentially provide increased amounts of genetic information.Item A Study of the Efficiency of the Combined DNA Index System for the Oregon State Police(2007-08-01) Brown, Allison A.; Joseph Warren; Arthur Eisenberg; John PlanzMethod of Procedure: This project, which was conducted at the Oregon State Police Crime Laboratory, entailed following up with CODIS hits. It involved examining cases that have been worked by forensic scientists and finding explanations as to how the cases are proceeding after NDA matches are made. Failures to follow up on a CODIS hit have become a national problem in forensic laboratories all over the country. The Oregon State Police find this a very important issue that must be resolved. Currently the Oregon State Police are getting a hit, an arrest and a conviction. The only problem with all of this is that it is hard to measure the efficiency of CODIS hits with just convictions. There are several ways the cases could have been resolved such as the victim did not want to pursue the case any further, the suspect was already incarcerated for another crime, the witnesses were hard to locate or the case was dropped because of a plea to other crimes. As part of my research I investigated each of the cases that have been worked to see if they were pursued any further after a hit to an individual. After researching these cases, it was my responsibility to put my findings into a format that made it easier for the state police to know how the cases were resolved. The information was collected using a variety of software programs. The reason that more then one computer program needed to be used in the project, is due to the fact that more of then not some of the information that should have been provided in a program was absent in one and present in another program. The California Department of Justice currently has a system that is available which allows its users to input data. In order to design such a system it was my responsibility to obtain the case information. Once this had been obtained it made it easier to combine the data into a table format so that the state police could see how each case was proceeding. Below is a table of exactly what information was obtained. Due to the confidentiality of the information, false names and false information will be used in all tables seen. Once the information for one thousand hits had been gathered it was placed into Microsoft Access so that a database could be created. Throughout the time that CODIS has been in place, crimes where DNA matches have been made but not pursued have shed light upon a common problem occurring everywhere in forensic laboratories. Dozens of cases have found matches between a suspect and a crime but there has been no pressure to pursue the case any further. In fact one unfortunate result has been multiple DNA matches of a suspect, with the suspect continuously making repeated offenses. Most of the offenses are nor pursued until the investigators realize there might be a link between the current crime and the past cases worked. Only then are the reports of the DNA matches reviewed and pursued further. In one Virginia case, this scenario occurred. A man by the name of William Orlando Smith followed a girl and raped her in the woods. Had the DNA match that the Virginia police made months earlier been pursued, the rape might have been prevented. This is not just one isolated event but a common occurrence seen in other states as well. Although this research for the Oregon State Police might not resolve a national issue, it will aid in preventing scenarios like the above from possibly occurring. By solving this problem and entering how cases are resolved into a database like Microsoft Access, it will make it easier for the investigators to process crimes. Even more important is the need for this information to be compared with other states. A study such as this will enable states to test the efficiency of CODIS.Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Allele Characterization of Fifteen Short Tandem Repeat Loci of North American Golden (Aquila Chysaetos) and Bald (Haliaeetus leucocephalus) Eagles using Next-Generation Sequencing(2014-05-01) Howard, Taylor E.; John PlanzBald and golden eagles are species of conservation concern in North America, and are protected under the Bald and Golden Eagle Protection Act (BGEPA; 16 U.S.C. 668-668d). Wildlife forensics utilizes short tandem repeat (STR) loci for identification purposes, however the loci currently used for bald and golden eagle in North America were developed from related species of European eagles. In this study, STR loci were sequenced using the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) to characterize the alleles (e.g. the repeat motifs, presence of SNPs and indels). These methods were used to evaluate the discriminatory power of the loci for individualization and for species differentiation.Item Allele Characterization of Ten Short Tandem Repeat Loci of North American Bears (Ursids) Using Next-Generation Sequencing(2014-05-01) Kreutzer, Mckensie; John PlanzAn identification method that provides higher genetic resolution than capillary electrophoresis (CE) is needed for isolated bear populations that possess low genetic diversity. Amplification conditions were optimized for ten bear STR loci. Amplicons were used to develop a library for next-generation sequencing (NGS) on the Ion Torrent™ PGM™ Sequencer. Through ligation of DNA barcode adaptors, seven black bear (Ursus americanus) samples were sequenced together. Sequencing reads were aligned to a virtual ladder and analyzed in NextGENe® software. Allele concordance was shown between CE and NGS. Variants within alleles (SNPs and INDELs) showed that NGS provided higher genetic resolution. These results have implications for improving individual identification and population assignment in wildlife forensics and conservation for populations with low genetic diversity.Item Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing(2004-08-01) Ariyo, Bolanle; Joseph Warren; John Planz; Arthur EisenbergAriyo, Bolanle. Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing Reactions. Master of Science (Forensic Genetics), August 2004, 46 pages, 10 figures, 7 tables, 18 references. Mitochondrial DNA is widely used in the forensic community because of its high copy number in cells, location, and mode of inheritance. Yet this method of analysis is expensive, time consuming, and labor intensive, therefore labs should take steps to improve the procedure of mtDNA analysis. This study is performed to validate the use of amplifying HVI and HVII region in its entirety (2 primer sets) for use in reference samples. Amplification performed using primers F15989-R16410 (HVI) and F73-R340 (HVII). The current method of amplification is 4 primer sets at full cycle sequencing reactions. The cost of Cycle Sequencing Kit is also expensive, therefore performing half and quarter reactions would be beneficial in reducing the amount of kit consumed. To validate the use of reducing cycle sequencing reactions, half and quarter cycle reactions were performed using 2 and 4 primer sets. Results demonstrate that sequence data for reducing cycle sequence data is consistent with the sequence data using the current method. Results also show that sequence data obtained using two primer sets was consistent with sequence data amplified by the current method with the exception of two samples at length heteroplasmy polyctosine regions.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 An Initial Comparison of Applied Biosystems Quantifiler Duo and Promega Plexor HY Real-time PCR DNA Quantification Systems(2008-05-01) Cole, Sarah Kathleen; Arthur Eisenberg; John Planz; Joseph WarrenObjective 1: Sensitive Study: This study was designed to determine the quantity of template DNA below which amplification is not expected to yield a DNA profile. Dilution series of male and female stock DNA ranging from 0.003 ng/μl will independently be run with both Quantifiler Duo and Plexor HY. These samples will be run in duplicate per plate, with duplicate plates being run. We want to determine if the published lowest detection thresholds (0.023 ng/μl for Duo; 0.0032 ng/μl for HY) are concordant with the data obtained. Objective 2: Mixture Study: The purpose of this study is to obtain quantification results for mixtures of male and female DNA, which should allow for calculations of autosomal:Y ratios that can be helpful in determining what type of genetic analysis to pursue (autosomal STR, Y-STR, or both). Mixtures of female and male DNA ranging from 1:1 to 1024:1 (female: male) will be run in duplicate per plate, with duplicate plates being run. We want to find out how minor of a contributor the male can be in an excess of female DNA and still be detected. This is especially important in sexual assault cases where the major contributor is usually female or when the offender is a vasectomized male. Objective 3: Concordance Study: The purpose of this study is to compare quantification results from Quantifiler Duo and Plexor HY with those from Quantifiler Human, specifically in cases when samples are degraded. The majority of these samples originate from unidentified human remains. Patterns of overestimation or underestimation of DNA concentration can help determine which system will be most beneficial in these cases. This is where the new amplicons size featured in Quantifiler Duo is important in comparing the values with previous results for Quantifiler Human. Sample choice will be at the discretion of the laboratory technical leader and Unidentified Human Remains section analysts. These samples will be the ones that are known to be degraded and have previously yielded overestimated results from the Quantifiler Human quantification system, resulting in poor STR data.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 Analysis of Low Copy Number DNA Using Profiler Plus at Increased Amplification Cycles and Modifications in Sample Injection Parameters(2003-08-01) Hynds, Jody Lynn; Arthur Eisenberg; John Planz; Joseph WarrenThere are many DNA testing techniques that can be utilized for samples with low quantities of DNA. Mitochondrial DNA testing is designed for successful DNA sequencing of hair shafts, degraded and burned samples. Newly developed SNP (single nucleotide polymorphisms) testing is also designed for the analysis of challenging samples. The increased interest in the analysis of low copy number DNA samples using STR testing is necessitated since the national database CODIS (Combined Data Index System) currently only accepts the DNA profiles analyzed with the 13 core STR loci. CODIS contains DNA profiles of evidence found at crime scenes, convicted offender and missing persons DNA profiles (4). The goal of this project is to develop methodologies to increase the success rate of LCN DNA samples using STR testing. The experimental design for this study involved the amplification of DNA isolated from buccal swabs using the Profiler Plus multiplex kit at two different DNA input quantities: 0/0156ng (15.6pg) and 0.0312ng (31.2pg). Four separate amplifications of these DNA samples were done at: 28, 30, 32 and 34 cycles. The manufacturer’s recommended cycle number for AmpFISTR Profiler Plus is 28 cycles. These samples were analyzed on both the ABI Prism 310 Genetic Analyzer and the ABI Prism 3100 Genetic Analyzer using OCD standard protocols for loading samples. The injection time and voltage were modified for each of the number of PCR cycles. The best combination of cycle number and injection parameters was chosen for the low copy number reproducibility study.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 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 Beta Testing and the Population Genetics of Promega's Prototype PowerPlex Y Kit(2004-08-01) Kirkendoll, Ross A.; Joseph Warren; John Planz; Arthur EisenbergDevelopmental validation is typically done by the manufacturer of the technique or technology. According to National Standards, the manufacturer must test for human specificity to ensure compliance with standards. In addition, the PowerPlex Y kit must be shown to have male specificity because all of the loci are located on the Y-chromosome. Other necessary studies include mixture both male/female and male/male mixture studies, stability studies to show stability in the presence of environmental insults, and the focus of this study the construction of a popular database. In order to satisfy both the requirement of the National Standards and the scrutiny of the legal system, Promega Corporation assembled a collaboration of different laboratories to assist with the developmental validation of the PowerPlex Y Kit. This project was a small part of that collaboration. The DNA Identify Laboratory was chosen by Promega to assist with the construction of a population database because of the number of samples available and the need for confirmed father/son pairs. The objectives of the study were to type ~200 father/son pairs from each of the Caucasian and African American races, and then determine the haplotype frequencies, haplotype diversities, and mutation rates for each race.Item Bone Morphogenetic Protein 4 inhibits TGF-beta2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation(2005-05-01) Zode, Gulab S.; Wordinger, Robert J.Zode, Gulab Shalikram, Bone Morphogenetic Protein 4 Inhibits TGF-β2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation . Doctor of Philosophy (Cell Biology and Genetics), May 2008; 177pp; 34 figures; bibliography, 192 titles. The glaucomatous neuropathy is caused by irreversible loss of retinal ganglion axons in the optic nerve head (ONH). The extensive remodeling of the extracellular matrix (ECM) in the glaucomatous ONH including increased synthesis and deposition of ECM (increased collagens, basement proteins, and elastin) is associated with loss of axons. Transforming growth factor-beta2 (TGF-β2) is increased in glaucomatous ONH and is thought to be responsible for increased synthesis and deposition of ECM proteins of the ONH. Bone morphogenetic proteins (BMPs) normally maintain the balance of ECM proteins via opposing TGF-β2 stimulated ECM proteins in various cell types. BMP antagonist gremlin inhibits BMPs function, thus may plan an important role in ECM modulation. We previously demonstrated that human ONH expresses BMP-4, BMP receptor and BMP antagonist gremlin. Therefore, we hypothesize that elevated TGF-β2 in the glaucomatous ONH induces gremlin expression that blocks BMP-4 inhibition of TGF-β2 signaling, leading to increased ECM synthesis and deposition. First, we examined whether human ONH tissues and ONH cells express the canonical BMP signaling pathway. This study demonstrated that ONH tissues and ONH cells express BMP-4 and Smad signaling pathway. Treatment of ONH cells with BMP-4 increased phosphorylation of R-Smad/1/58/ phosphorylation and interaction with Co-Smad4 indicating activation of the Smad signaling pathway. Therefore, cells within the human ONH can respond to locally released BMP via activation of Smad signaling. Second, we examined the signaling pathways utilized by TGF-β2 to stimulate ECM in ONH cells. This study demonstrated that TGF-β2 is increased in glaucomatous ONH. Recombinant TGF-β2 increased ECM deposition in ONH cells. TGF-β2 activated phosphorylation of R-smad2/3 but did not alter phosphorylation of ERK1/2, p38, and JNK1/2 in ONH cells. Inhibition of either TGF-β I receptor activity or phosphorylation of R-Smad3 or knockdown of R-Smad2/3 via siRNA reduced TGF-β2 stimulated ECM in ONH cells. Thus, TGF-β2 requires R-Smad2/3 to stimulate ECM proteins in ONH cells. Lastly, we investigated the potential effects of BMP-4 and gremlin on TGF-β2 stimulated ECM in ONH cells. BMP-4 significantly reduced TGF-β2 stimulation of ECM proteins. Addition of gremlin blocked the BMP-4 effect, increasing ECM proteins in ONH cells. Gremlin levels were significantly increased in the human glaucomatous ONH tissues. Interestingly, recombinant gremlin also increased ECM proteins in ONH cells. Gremlin stimulation of ECM proteins required activation of the TGF-β receptor and R-Smad3. TGF-β2 increased gremlin mRNA and protein in ONH cells. Thus, TGF-β2 induced gremlin expression intensifies TGF-β2 effects on ECM metabolism by inhibiting BMP-4 antagonism of TGF-β2 signaling. In conclusion, elevated TGF-β2 and gremlin in the glaucomatous ONH are involved in the pathogenesis of glaucomatous ONH. Elevated TGF-β2 directly increases ECM and also induces gremlin expression, which further aids TGF-β2 to stimulate ECM via inhibiting BMPs antagonism of TGF-β2 signaling, leading to unopposed TGF-β2 stimulated ECM proteins. Interestingly, R-smad3 is required for TGF-β2 or gremlin induced ECM remodeling in ONH cells. Therefore, modulation of R-smad3 provides a novel therapeutic target for preventing ECM remodeling in glaucoma.Item Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways(2003-05-01) Taylor, Sara A.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouTaylor, Sara A., Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways. Master of Science (Biomedical Sciences), January, 2003, 112 pp., 4 tables, 39 illustrations, bibliography, 68 titles. Purpose: Glutamate has been implicated in many pathologies affecting the Central Nervous System including those in the retina, but the exact nature of the role of glutamate in neuronal degeneration remains unclear. In the retina. Müller cells are resistant to glutamate insults that are normally toxic to other cells of the retina, however the molecular and biochemical mechanisms that control their death or survival are not well understood. We used a series of pharmacological inhibitors and molecular biology agents on cultured Müller cells to dissect two key signaling pathways normally involved in cell survival, the Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) pathway and the Phosphatidylinositide 3 Kinase (PI3K) pathway. Since preliminary data in our laboratory showed that Müller cells upregulate their secretion of neurotrophins including Brain Derived Growth Factor (BDNF) in response to glutamate treatment, we also examined the effect of BDNF on the activation of these two signaling pathways. Methods: Early passaged Müller cells were treated with various concentrations (5 nM -50 μM) of inhibitions of the MAPK(ERK) pathway (GW5074, U0126, and PD98059) or with various concentrations (1-50 μM) of inhibitors of the PI3K pathway (LY294002 or Akt inhibitor) in the presence and absence of 50 ng/ml of BDNF for 24 hours. These experiments were repeated in Müller cells transfected with either NFκB or Bc12 DNA. Cell cultures were then analyzed for surviving cells with an MTS/PMS assay, a colorametric method for determining the number of viable cells in a proliferation assay. Results: The MAPK (ERK) inhibitors PD98059 and GW5074 both resulted in decrease in Müller cell survival. PD98059 did not decrease Müller cell survival until concentrations were high enough to suppress ERK2 phosphorylation. Müller cells transfected with NFκB or Bc12 DNA were able to resist treatment with concentrations of PD98059 that reduced cell number in untransfected cells. The PI3K inhibitor LY294002 also resulted in significant decreases in Müller cell survival in both untransfected cells and cells transfected with NFκB or Bc12 DNA. Treatment with an inhibitor farther down in the PI3K pathway, Akt inhibitor, did not significantly decrease Müller cell survival. Finally, BDNF was not able to increase cell survival in Müller cells treated with PD98059 or U0126, although it did increase the survival of cells treated wit GW5074. BDNF was also able to reverse the decrease in cell survival caused by LY294002 in both untransfected Müller cells or Müller cells transfected with NFκB or Bc12 DNA. Conclusions: Our data shows that Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) and Phosphatidylinositide 3 Kinase (PI3K) are both essential for Müller cell survival. There is modulation between the pathways and they may interconnected far upstream at a protein previously associated with only the MAPK(ERK) pathway. These results are consistent with a role for both pathways in Müller cell survival.