Browsing by Subject "Genomics"
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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 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 Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism(2002-05-01) Campbell, Rowan Stewart; Arthur J. Eisenberg; Bruce Budowle; John PlanzCampbell, Rowan Stewart, Automatable Virtual Array Screening System For Rapid Analysis of Mitochondrial DNA Polymorphism. Doctor of Philosophy (Biomedical Sciences), May, 2002, 156 pp., 11 tables, 48 illustrations, bibliography, 96 titles. The goal of this research project was to develop alternative methods to traditional forensic mtDNA sequence analysis. Conventional forensic mtDNA analysis requires the direct sequencing of Hypervariable Region I and Hypervariable Region II in both the forward and reverse directions. This method is time consuming, labor intensive and expensive. Two methods for determining mtDNA haplotypes through the direct interrogation of Single Nucleotide Polymorphisms with HVI and HVII have been developed. A Sequence Specific Oligonucleotide Hybridization assay was developed on the Luminex 100™ flow cytometer, as well as a Single Base Extension assay developed for the ABI Prism® 310 Genetic Analyzer. The SNP typing of mtDNA sequences can provide a significant benefit in many forensic and human identification cases. The reassociation of mass disaster remains, mass grave analysis, and the screening of large numbers of crime scene samples are examples of their potential application. Their inclusion as a standard screening tool would be high beneficial since more extensive DNA analysis would be reserved for those samples that possess the greatest evidentiary value. In a blind study of 50 samples, the Sequence Specific Oligonucleotide Hybridization assay incorrectly identified the mtDNA haplotypes in 7 samples, whereas the Single Base Extension assay correctly identified each of the SNP positions interrogated. The SNaPshot™ primer extension assay was approximately 20-25 times more sensitive than the standard sequencing approach. This would suggest that this system could be a viable alternative to sequence analysis when samples are limited, as well as being more robust in detection and typing of heteroplasmic sites. A statistical evaluation of the SNP panels revealed that the genetic diversity estimated for the 50 Southwestern Hispanic samples tested was 0.9624 for the primer extension array and 0.9559 for the hybridization-based array. The probability of two randomly selected individuals from a population group having the same mtDNA haplotype was 0.0568 for the Single Base Extension assay and 0.0632 for the Sequence Specific Oligonucleotide Hybridization assay. A forensic mtDNA SNP array consisting of the positions evaluated in this study could provide a reasonable alternative to the full sequencing of the HVI and HVII regions.Item Changes in Mammalian Chromatin Structure as a Function of Protein-Poly(ADP-Ribosyl)ation by Endonuclease Digestion(2004-06-01) Perez-Lamigueiro, Maria A.; Alvarez, Rafael; Das, Hriday K.; Basu, AlakanandaPerez-Lamiguerio, Maria A., Changes in Mammalian Chromatin Structure as a Function of Protein-poly(ADP-ribosyl)ation by Endonuclease Digestion. Master of Science (Biochemistry and Molecular Biology), June 2004. 66 pages, 12 illustrations, Bibliography, 45 titles. Mammalian chromatin was exposed to either Deoxyribonuclease I or Micrococcal Nuclease digestion as a function of time of incubation and enzyme concentration. Endonuclease enzymatic reactions were stopped with EDTA. Samples were run in 1.5% agarose gels and the oligonucleosomal electrophoretic migration patterns compared. Endonuclease experiments were carried out with rat liver chromatin pre-incubated in the presence or absence of 200 μM βNAD+. A solution of 1.0 mM benzamide was used to stop enzymatic modification. The electrophoretic observations demonstrated a faster and increased degradation of chromatin when proteins were poly(ADP-ribosyl)ated prior to digestion. These results support the hypothesis that that the covalent poly(ADP-ribosyl)ation of chromatin proteins, particularly histones, induces a more relaxed structure, rendering chromatin more sensitive to endonuclease digestion.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Differentiation of Vascular Smooth Muscle Cells(2004-05-01) Deaton, Rebecca Ann; Dillon, Glenn; Shepard, Allan; Mallet, Robert T.Rebecca Ann Deaton, Characterization of the role of PKN in TGF-beta 1-mediated differentiation of vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), May 2004, 178 pp, 5 tables, 34 illustrations, references, 197 titles. Differentiated vascular smooth cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors. SMCs retain the ability to de-differentiate into an “immature” proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanism involved in maintain SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-B1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-B1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs. These characteristics are indicative of the differentiated phenotype. The effects of TGF-B1 can be blocked by pretreatment of the cells with either HA1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-B1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM a-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF-GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-B1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK ½. Co-expression of dominant negative p38 MAPK is sufficient to abolish PNK-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-B1 activates RhoA and PKN to promote differentiation of SMCs.Item 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.Item Dr. Arthur Eisenberg(2013-08-01) Eisenberg, Arthur J.; Milligan, JessieItem Dr. John Planz(2013-08-01)John V. Planz holds a B.S degree in Biology and Zoology (double major) from the State University of New York (Oswego, NY), a M.S. degree in behavioral ecology from Shippensburg University (Shippensburg, PA) and a Ph.D. in molecular evolutionary genetics and population genetics from the University of North Texas (Denton, TX). Dr. Planz studied as a postdoctoral fellow at the Carnegie Museum of Natural History, Section of Mammals (Pittsburgh, PA) in mammalian phylogenetic systematics. Dr. Planz entered the forensics field in 1993 at the Southwestern Institute of Forensic Sciences in Dallas, TX. He later served as the Director of Identity Testing at GeneScreen, Inc. in Dallas, TX and Biosynthesis, Inc. in Lewisville TX adding mitochondrial DNA typing and SNP development to the testing performed by those laboratories. Dr. Planz joined the faculty of the University of North Texas Health Science Center at Fort Worth in January 2000 were he serves as an Associate Professor in the Department of Forensic and Investigative Genetics and is the Associate Director of the UNT Center for Human Identification.Item Dr. Joseph Warren(2013-08-02)Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.Item How do you get a job in Forensic Science?(2013-08-01)Item Identification of Novel Genes Involved in Escherichia coli Biofilm Formation(2003-05-01) DesPlas, Rebecca L.; Jerry Simecka; Ming-Chi WuDesPlas, Rebecca L., Identification of Novel Genes Involved in Escherichia coli Biofilm Formation. Master of Science (Microbiology), May 2003, 77 pp., 6 tables, 11 figures, references, 55 titles. Transposon mutagenesis using a miniTn10::camR transposon generated 800 random insertion mutants displaying altered biofilm phenotypes as compared to the parent strain, TRMG F/M. Transduction of the resistance marker confirmed approximately 150 biofilm mutants. Amplifications of the insertion sites, nucleotide sequencing and BLAST searches against E. coli K-12 genomic databases, identified118 of these sites. Many of the interrupted genes are not known to be associated with biofilm formation. Four mutations were transduced into E. coli K-12 MG1655, creating altered biofilm phenotypes. A plasmid clone of the nhaAR operon complemented the corresponding mutations. Results indicate that the genes identified in this study influence biofilm formation. However, further studies are needed to determine the degree of impact in a wild type strain background.Item Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, ZygmuntAdkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.Item Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning(1996-07-01) Price, Nancy E.; Neeraj Agarwal; Robert Easom; Stephen R. GrantPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.Item Molecular Characterization of a Second Superoxide Dismutase Gene (sodM) of Staphylococcus Aureus and Effects of SodM and SodA on Oxidative Stress Resistance and Virulence(2001-11-01) Valderas, Michele Wright; Porunelloor A. Mathew; Richard Easom; Jerry SimeckaValderas, Michelle Wright., Molecular Characterization of a Second Superoxide Dismutase Gene(sodM) of Staphylococcus aureus and Effects of SodM and SodA on Oxidative Stress Resistance and Virulence. Doctor of Philosophy (Biomedical Sciences), November, 2001, 191 pp., 4 tables, 20 illustrations, bibliography, 389 titles. A second gene for superoxide dismutase (SOD) in S. aureus (sodM) was cloned and characterized. This gene was found to be unique to S. aureus among the staphylococci. S. aureus is the first gram-positive bacterium reported to contain two or more SOD activities. The three native SOD enzymes observed for S. aureus can be accounted for by two distinct genes, sodM and sodA. The SodM and SodA proteins form homodimers, but their subunits also interact to form an active heterodimeric SOD. The deduced amino acid sequence from each gene and the relative insensitivity to hydrogen peroxide and potassium cyanide indicated that the S. aureus SODs utilize manganese as a metal ion cofactor. Additionally, viabilities of the soda and sod double mutants, but not the sodM mutant were drastically reduced under conditions of oxidative stress in early exponential growth. However, only the double mutant was affected when oxidative stress was applied in the late-exponential and stationary phases of growth. It was determined, therefore, that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under conditions of oxidative stress, becomes a major source of SOD activity during the late-exponential and stationary phases of growth such that the viability of the S. aureus sodA mutant is maintained. Experiments examining the roles of sodM and sodA in virulence determined the ability of S. aureus to cause disease in the mouse lung and subcutaneous abscess formation in mice was unaffected by sod mutation. Lack of SOD activity, however, results in enhanced clearance of S. aureus within the lung and also promotes killing of the organism by mouse macrophage cell lines and human polymorphonuclear leukocytes.Item Molecular Cloning and Regulation of Expression of an NK Cell Receptor(2001-07-01) Medina, Miguel Angel; Porunelloor Mathew; Rafael Alvarez-Gonzales; Neeraj AgarwalNatural killer (NK) cells are large granular lymphocytes derived from bone marrow. They form the first line of defense against virally infected and tumor cells. Unlike B and T cells, they are not MHC restricted therefore do not require prior antigen stimulation (1-4). NK cell functions include producing various cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and granular-macrophage colony stimulating factor (GM-CSF) and cytotoxicity (5,6). A number of cell surface molecules have been identified, cloned and characterized that modulate NK cell recognition and activation by target cells (1). Most of these molecules are also expressed on other leukocytes. NK cell function is regulated by the balance of the positive and negative signaling through these receptors (3, 7-10). In the past attention has primarily focused on major histocompatibility complex (MHC) recognizing receptors that are mostly inhibitory (11). It is through these inhibitory receptors that levels of MHC molecules and associated peptides are monitored. Cells that have lost the expression of MHC class I molecules or have altered peptides-class I complexes are not able to transmit an inhibitory signal to NK cells and are consequently killed. Members of the CD2 subset of receptors play a major role in lymphocyte functions and do not recognize MHC molecules. The signaling lymphocyte activation molecule, SLAM (CD150), a member of the CD2 subset, is expressed on T cells and B cells. SLAM regulates T cell activation and production of immunoglobulins by B cells (12,13). 2B4 is a member of the CD2 subset and is expressed on NK cells as well as other leukocytes (14, 15). 2 B4 is a surface molecule implicated in the activation of NK cell-medicated cytotoxicity (15-17). Human 2B4 is a 60-70 kDA glycoprotein surface molecule found on all NK cells and a small subset of T cells that exhibit NK-like activity. CD48 has been identified as the high affinity ligand for 2B4 and implicates a broader role for 2B4 in immune regulation (18, 19). Recent reports have demonstrated the importance 2B4 and the functional role 2B4 plays in immune regulation. In X-linked lymporoliferative (XLP) disease NK cells can not be activated through surface 2B4 (20-23). The molecular adaptor protein, SLAM-associated protein or SH2 domain containing adaptor molecule (SAP/SH2D1A) is associates with cytoplasmic tail of 2B4 or SLAM (24, 25). Defective signaling via 2B4 and SLAM may contribute to the pathogenesis of X-linked lymphoproliferative disease due to mutations in SAP. The cytoplasmic domain of 2B4 contains four novel tyrosine motifs (TxYxxV/I) (14, 15). SLAM, a close relative of 2B4, also contains these novel tyrosine motifs. The signaling mechanism for 2B4 remains unclear. Along with other members of the CD2 subset 2B4 also localizes to chromosome 1. The genes that encode the CD2 family of receptors are locatedon human chromosome at 1q21-24 (24, 26-30). The murine genes for 2B4, CD48, Ly49, Ly108, and CD84 are located on the syntenic region of the long arm of the chromosome 1 (30-33). The exon arrangement for 2B4 is consistent with other CD2 subset members and consists of an exon per domain for the leader sequence, V-like domain, C2-like domain, and the transmembrane domains (27, 34-37). Differential exon usage leads to splice variants of the receptors, which complicates understanding the functional relevance between the cytoplasmic domains between receptors. Both murine 2B4 and SLAM demonstrates splice variants that alter the number of novel tyrosine motifs within the cytoplasmic domains (14, 34, 38). The murine 2B4 gene consists of 9 exons with one exon dedicated to each leader sequence, V-like, C2-like, and transmembrane domains. The total gene size is approximately 27 kilobases with the first intron consisting of 16 kilobases. Variable exon usage gives rise to two isoforms of 2B4, 2B4-L and 2B4-S, in the mouse (38). Four exons encode the 2B4-L cytoplasmic domain, giving rise to four tyrosine motifs. 2B4-S is identical to the 5’end of 2B4-L, differing only at the 3’ end in a portion of the cytoplasmic domain and the 3’untranslated sequence. 2B4-S is the product of the same first five exons in 2B4-L with the usage of a novel exon at the C-terminal. Although splice variants exists there Is no direct biochemical evidence to support their expression. In vitro analysis of the m2B4 variants suggest potential signaling differences. Murine 2B4 variants and mutants were transfected into a rat NK cell line, RNK-16. Interestingly, the two forms of 2B4 had opposing functions (39). Murine 2B4 is expressed on all NK cells, a subset of T cells, dendritic epidermal T cells, and monocytes (40). Expression levels of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 with anti-2B4 monoclonal antibody (mAb) causes secretion of interferon-γ, increased 2B4 expression, and elevated cytotoxicity (41). Characterization of how 2B4 and its related receptors are expressed is critical to the understanding not only the receptors’ biology but also NK cell biology. My first project will focus on mastering the techniques involved in the isolation and characterization of genes. Previously two genomic clones were isolated from 129 Sv/J mouse liver, 531 and 532. The first clone, 531, has been fully characterized and revealed to be 2B4. 532 has been partially characterized and revealed to the related form of mouse 2B4. In order to determine the function of 532 on mouse NK cells, 532 cDNA has to be isolated. I attempted to isolate 532 cDNA through PCR using previously isolated clones from the BALB/c cDNA library. My next aim was to isolate 532 genomic DNA for automated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated response molecule. The final portion of my thesis will focus on the isolation of the 2B4 activated response molecule (2ARM). Human peripheral blood NK cells were isolated incubated with interleukin-2 or C1.7. C1.7 is a monoclonal antibody that specifically recognizes human 2B4. RNA was extracted from these NK cells at various time points and used for RT-PCR to monitor the expression levels of 2B4. Aside from the expression of human 2B4, the expression of a 160 base pair transcript was also detected. Sequencing analysis revealed this transcript to be novel. I screened a human NK cDNA library constructed by Dr. J. Houchins (R & D System, Minneapolis, MN) using this 160 base pair transcript as a probe. Upon isolation of 2ARM cDNA, functional analysis can be performed to determine its role on human NK cells.