Browsing by Subject "Genetic Structures"
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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 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 Adaptation of the Genetic Risk Prediction Model BRCAPRO for Primary Care Settings(2017-05-01) Atienza, Philamer M.; Sumihiro Suzuki; Swati Biswas; Karan P. SinghIdentifying women at high risks of carrying the breast cancer susceptibility genes is crucial for providing timely surveillance and necessary health management interventions. BRCAPRO is one of the most widely used statistical models for breast cancer risk prediction in genetic counseling. It provides carrier probabilities of BRCA1/2 mutations and calculates the risks of developing breast and ovarian cancers. This calculation requires extensive personal and family history information, which makes it difficult to use in primary care where a wider population could be reached. Thus, we developed a two-stage approach for the genetic risk prediction of BRCA1/2 mutation. In the first stage, limited information on the counselee and her family history of cancer are used in simplified versions of BRCAPRO. If the risk at this stage is found to be high, the full BRCAPRO model utilizing the complete family history is implemented in the second stage. We aimed to balance the tradeoff between the amount of information used and the accuracy of the predictions. We explored several first stage tools. BRCAPROLYTE uses information on the affected relatives up to the second degree only. BRCAPROLYTE-Plus additionally includes unaffected relatives by imputing their ages. BRCAPROLYTE-Simple eliminates the need to collect information on the numbers and types of unaffected relatives and imputes them and their ages instead. The study cohorts include 1,917 families mostly at high risk from the Cancer Genetics Network, 796 high-risk families from MD Anderson Cancer Center, and 1,344 population-based families from Newton-Wellesley Hospital. To evaluate the models, we used sensitivity, specificity, area under the curve, and observed versus expected number of carriers. We also considered clinical criteria of number of referrals made by each model. We found the proposed two-stage approach (with BRCAPROLYTE, BRCAPROLYTE-Plus, and BRCAPROLYTE-Simple at the first stage) has very limited loss of discrimination and comparable calibration with BRCAPRO. It identifies a similar number of carriers without requiring a full family history evaluation on all probands. Thus, our two-stage approach allows for practical large-scale genetic risk assessment in primary care.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 mitochondrial genome sequencing using the oxford nanopore minion device(2017-05-01) Thorson, Kelcie C.; John V. Planz; Rance E. Berg; J. Thomas CunninghamCurrent DNA sequencing methods rely on polymerase chain reaction (PCR) to create sufficient copies of targeted DNA fragments to serve as a library. PCR and subsequent clean-up steps add considerable time and cost to the process and provide opportunity for introduction of amplification errors or contamination. The aim of this study was to develop a reliable method for mitochondrial genome sequencing sans PCR using the Oxford Nanopore MinION. Our hypothesis was that results generated from native DNA sequencing would be concordant to sequencing results generated from PCR-enriched libraries. Error-rates of 1.47% (enriched) and 1.26% (native) were observed when sequencing the control DNA. Consensus sequences generated from native and PCR-enriched libraries show an average of 98.7% identity between the two methods.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 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 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 DNA Extraction Methods From Bone to be Used With the DNA IQ System on the Maxwell 16 for Human Identification(2008-08-01) Lopez, Kristen; John Planz; Arthur Eisenberg; Joseph WarrenLopez, Kristen M., Comparison of DNA Extraction Methods From Bone to be Used with the DNA IQ System on the Maxwell 16 for Human Identification . Masters of Science (Graduate School of Biomedical Sciences), August, 2008, 52 pp., 11 tables, 15 figures, bibliography, 24 titles. Extraction and purification of DNA from human bones is essential for correctly identifying the remains through DNA analysis. Current DNA extraction methods include a demineralization step, which extracts calcium and phosphate from the bone matrix, inactivation of DNAses, and the removal of Polymerase Chain Reaction (PCR) inhibitors. These methods often use harsh chemicals and may allow for residual DNA to be discarded in various wash steps. To assess the effectiveness of DNA extraction from bone samples, two extraction protocols were compared. The first method included a bone demineralization pretreatment solution of Sodium N-Laurylsarcosinate, 0.5 M EDTA, and Proteinase K (20 mg/ml). The second included a pretreatment using a Bone Incubation Buffer by Promega Corporation, with an addition of Proteinase K (18mg/ml). Various incubation times were included to assess the extraction at different time intervals. All extracted samples were purified with the DNA IQ Reference Sample Kit on the automated Maxwell 16 Instrument (Promega Corp.). Full and partial profiles were obtained from samples extracted with the Bone Incubation pretreatment, regardless of incubation time. Profiles were not observed with the standard demineralization pretreatment when amplified at 28 cycles, with partial profiles present in a few samples when amplified at 32 cycles.Item Concordance Study of Forensic Casework Samples Using the AMPFlSTR Kit, AMPFlSTR Identifiler Kit, AMPFlSTR Profiler Plus Kit and PowerPlex 16 Kit(2002-07-01) Armstrong, Treva L.; Arthur Eisenberg; John Planz; Joseph WarrenThe PowerPlex 16, AmpFlSTR Profiler Plus and AmpFlSTR COfiler Kits allow for the co-amplification of the amelogenin gender determining marker and the thirteen core CODIS STR loci: D3S1358, FGA, vWA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1, TPOX and CSF1PO. PowerPlex 16 adds the Penta D and Penta E loci and the AmpFlSTR Identifiler addes the D2S1338 and D19S433 loci. Manufactures of these systems have a suggested input DNA sample range of 0.5-2.5 ng, but have been successfully used to type samples containing less than 0.5 ng of DNA. In this study, several questions were addressed: First, “Are all four STR multiple kits concordant in their reproducibility, reliability, sensitivity and efficiency?” Second, “Is one particular STR megaplex kit more applicable to routine forensic casework?” and Third, “In a mixed DNA sample can individuals, whether male or female, be differentiated?” This paper describes a casework concordance study using adjudicated nonprobative sexual assault, mixed DNA and reference blood samples. Amplifications on all samples, were performed using the AmpFlSTR COfiler, Profiler Plus and Identifiler and PowerPlex 16 Kits and genotyping results were obtained using GeneScan and Genotyper software.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 Crime Scene Investigation: TV versus Reality(2013-08-01) ; Warren, Joseph; Budowle, Bruce; Eisenberg, Arthur J.; Pullin, Mike; Milligan, JessieJoseph Warren, Bruce Budowle and Arthur J. Eisenberg speak about crime scene investigation and forensic science as portrayed in popular television. They discuss how the shows distort and overstate the ways in which forensic scientists help solve crimes and identify victims, and they describe potential impacts on jurors' expectations. They also appreciate how these shows drive curiosity and bring better grant funding and more students to forensic science.Item Development of a Comprehensive Massively Parallel Sequencing Panel of Single Nucleotide Polymorphism and Short Tandem Repeat Markers for Human Identification(2015-08-01) Warshauer, David H.; Bruce Budowle; Ranajit Chakraborty; Bobby L. LaRueMassively parallel sequencing (MPS) technologies allow for the detection of an unparalleled amount of genetic information with unprecedented speed and relative ease. These qualities make the technology desirable for generating DNA profiles that may be uploaded into forensic offender, arrestee, and family reference database files. This doctoral dissertation research was conducted under the hypothesis that MPS, with its exquisitely high throughput, can provide a system whereby reference samples can be typed for a large battery of markers, providing more discrimination power for forensic DNA typing and offering increased opportunities to develop investigative leads. The design and implementation of large marker panels for the typing of reference samples will reduce debates on the best core markers for forensic utility, generate innovation because focus will not be solely on a core set of autosomal STRs, promote the development of better systems that can analyze more challenging samples, and enable sharing of data across laboratories worldwide. The primary goal of this project was to develop the capability of typing reference samples for a large battery of markers: 84 autosomal, Y-chromosome, and X-chromosome short tandem repeats (STRs), Amelogenin, and 275 human identity single nucleotide polymorphisms (SNPs), in a single multiplex analysis. To that end, a bioinformatic software package, STRait Razor, was developed to detect STR alleles in raw MPS data. A proof-of-concept study was performed to evaluate the efficacy of using MPS to type forensically relevant markers, using a PCR multiplex-based SNP assay. The proposed comprehensive capture-based MPS panel then was designed and extensively tested. Finally, the benefits of the additional genetic data afforded by MPS, as opposed to traditional methods, were illustrated through the characterization of intra-repeat nucleotide variation within Y-chromosome STR alleles. The results of this dissertation research indicate that MPS is capable of providing robust genetic data from a wide variety of forensically-relevant STR and SNP loci in a single analysis. To date, the comprehensive MPS panel developed during the course of these studies is the most potentially informative assay for reference sample testing for human identification.Item Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure(2006-06-01) Selby, Donald Evan; Stephen R. Grant; Patricia A. Gwirtz; Dan DimitrijevichDonald Evan Selby, Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure. Doctor of Philosophy (Biomedical Sciences), July 2006, 235 pp, 4 tables, 35 illustrations, references, 328 titles. Pacing induced tachycardia (PIT), in mammals, is known to cause a change from normal heart function to early left ventricular dysfunction. Progression to heart failure in experimental animals, such as dogs, pigs, and sheep, takes place in a relatively short period of time compared to the disease development observed in humans. Due to the cost and nature of using such animals, there is a need for a small animal model of PIT, which would delineate the etiology of the disease state by impairing the systolic function. The mode of action of overpacing inducement of cardiomyopathy, as the data suggests, may be through a sarcomere stretch sensor and its length-dependent signaling mechanism. In this study, an internal electrical-overpacing of an isogenic rabbit strain over a 52-day period was used to initiate a pathology consistent with human CHF. The data presented demonstrated that PIT causes alterations in the systolic ability of the heart, observed as reduced fractional shortening of the heart. This is seen in changes of the message pool population for proteins of the contractile architecture. Initially the heart is being paced rapidly and therefore there is insufficient time to get blood into the chamber. Thus, the data suggests that a mechanical stretch sensor is the process by which overpacing the heart leads to changes in gene expression which ultimately cause a compounding cellular condition which exists during heart failure. The data shows that there are gene isoform ratio changes that occur as the disease develops these include changes in differential expression of cardiac titin alternative splicing isoforms. The data suggests that there is also isoform switching occurring with alternative splicing of the gene encoding for SERCA2a, the probe 1587641_at shows a moderate decrease in expression and using BLAST for this probe this sequence is homologous to an alternative splicing variant of SERCA2a of the rabbit accession number J04703. The data shows that ferritin heavy chain also has an alternative splicing variant that are differentially regulated, this dysregulation of the isoform ratio may be linked to ADAMSTS1, a disintegrin and metalloproteinase isoform 1, which is seen to be downregulated in the data, these play a role in negative regulation of cellular proliferation. In addition to these detected isoform changes in the ratios of alternative splice variants changes are seen in genes linked to sarcomere integrity such as dystrophin probe 1582958_at is significantly increased in its expression, also integrin beta-1 probe 1584175)at shows a marginal increase in expression. The protease calpain probe 1604384)at, which uses a substrate the aforementioned integrin, dystrophin, and titin is also significantly upregulated in the data. Interestingly calpastatin, probe 1591603_at the inhibitor of calpain is marginally increased in its expression. Only recently has titin become to be appreciated as the protein that is responsible for the Frank Starling law as it undergoes an isoform ratio change as heart failure develops. These changes are initially caused by changes in ion concentration and stress upon the contractile proteins but as seen in the study, leads to altered gene expression. In this model, these gene alterations lead to diastolic dysfunction and the compounded problems constitute heart failure. This work shows that heart failure induced by over-pacing creates physical demands upon the framework of the heart and these physical stresses are transmitted through mechanical sensors leading to differential expression of the message pools for proteins involved in the way the heart contracts, and fills upon relaxation which ultimately ends in a heart that can do neither, thus leading to death.Item DNA-Prokids: Forensic Genetics and Human Trafficking(2013-08-01) Eisenberg, Arthur J.DNA - ProKids is a program jointly run by the Universidad de Granada (UGR) and the University of North Texas Health Science Center (UNTHSC). The Goal of DNA - Prokids is to return trafficked individuals, specifically young people and children to their families in their home countries. The FBI estimates that there are more than 100,000 children and teens in the United States that are being trafficked in the sex trade. Since Texas is a portal into the U.S. for Central and South America, the state government has been proactive in trying to address the problem. Part of the solution is the partnership between UNTHSC and UGR to work on DNA-Prokids. DNA-Prokids is an international humanitarian effort to help identify missing children and if possible to reunite abducted and homeless children with their parents, and to provide law enforcement agencies a scientific methodology to help deter the human trafficking of children. The organization also works with law enforcement to ensure that the children are returned to their families or placed in safe environments. Since this video, 697 parent-child associations have been made, with the vast majority of those children being returned to their families. Out of those 697, approximately 12 children have not been able to return home due to crime, violence, and drugs in their biological families. DNA-Prokids has also detected and avoided 221 illegal adoptions, where the woman presenting the baby to be adopted was not the child's biological mother. The success of the program deters human trafficking and can prevent child abuse and slave labor by increasing coordination between efforts like DNA-Prokids and authorities.