To see the other types of publications on this topic, follow the link: DNA replication – Research – Methodology.
Dissertations / Theses on the topic 'DNA replication – Research – Methodology'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 33 dissertations / theses for your research on the topic 'DNA replication – Research – Methodology.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Wendel, Brian Michael. "Completion of DNA Replication in Escherichia coli." PDXScholar, 2018. https://pdxscholar.library.pdx.edu/open_access_etds/4406.
Full textAbstract:
To maintain genomic integrity, all cells must accurately duplicate their genetic material in order to provide intact and complete copies to each daughter cell following cell division. Successful inheritance of chromosomal information without changing even a single nucleotide requires accurate and robust DNA replication. This requires that cells tightly control replication initiation from the origin(s), processive elongation of the replisome, and the completion of DNA replication by resolving convergent replication forks ensuring that each sequence is duplicated without alteration. Unlike initiation and elongation, the process by which replication forks converge and are resolved into two discrete, inheritable DNA molecules is not well understood. This process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is likely to be a fundamental step in regulating genome stability in all cells.
In this dissertation I address how DNA replication completes in the model system Escherichia coli. To achieve this, I examined candidate mutants for impairments in the completion of DNA replication. By evaluating growth, viability, chromosomal copy number, and plasmid stability I identified a requirement for the proteins RecBCD, ExoI, and SbcCD in the completion reaction. SbcCD and ExoI act before RecBCD in the completion reaction and process the DNA intermediates arising as replication forks converge. These enzymes act in the completion reaction without recombination or RecA, but in the absence of the normal process recombination is required to complete DNA replication via an aberrant pathway that results in genomic instability.
2
Hamilton, Nicklas Alexander. "Use of Two-replisome Plasmids to Characterize How Chromosome Replication Completes." PDXScholar, 2019. https://pdxscholar.library.pdx.edu/open_access_etds/5064.
Full textAbstract:
All living organisms need to accurately replicate their genome to survive. Genomic replication occurs in three phases; initiation, elongation, and completion. While initiation and elongation have been extensively characterized, less is known about how replication completes. In Escherichia coli completion occurs at sites where two replication forks converge and is proposed to involve the transiently bypass of the forks, before the overlapping sequences are resected and joined. The reaction requires RecBCD, and involves several other gene products including RecG, ExoI, and SbcDC but can occur independent of recombination or RecA. While several proteins are known to be involved, how they promote this reaction and the intermediates that arise remain uncharacterized.
In the first part of this work, I describe the construction of plasmid "mini-chromosomes" containing a bidirectional origin of replication that can be used to examine the intermediates and factors required for the completion reaction. I verify that these substrates can be used to study the completion reaction by demonstrating that these plasmids require completion enzymes to propagate in cells. The completion enzymes are required for plasmids containing two-replisomes, but not one replisome, indicating that the substrate these enzymes act upon in vivo is specifically created when two replication forks converge.
Completion events in E. coli are localized to one of the six termination (ter) sequences within the 400-kb terminus region due to the autoregulated action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner. In the second part of this work, I examine how the presence of ter sequences affect completion on the 2-replisome plasmid. I show that addition of ter sequences modestly decreases the stability of the two-replisome plasmid and that this correlates with higher levels of abnormal, amplified molecules. The results support the idea that ter sites are not essential to completion of DNA replication; similar to what is seen on the chromosome.
Rec-B-C-D forms a helicase-nuclease complex that, in addition to completion, is also required for double-strand break repair in E. coli. RecBCD activity is altered upon encountering specific DNA sequences, termed chi, in a manner that promotes crossovers during recombinational processes. In the third part of this work, I demonstrate that the presence of chi in a bidirectional plasmid model promotes the appearance of over-replicated linear molecules and that these products correlate with a reduced stability of the plasmid. The effect appears specific to plasmids containing two replisomes, as chi on the leading or lagging strand of plasmids containing one replisome had no-effect. The observation implies chi promotes a reaction that may encourage further synthesis during the completion reaction, and that at least on the mini-chromosomes substrates, this appears to be a destabilizing force.
3
Godfrey, D. B. "Cloning of a gene involved in replication of DNA on a damaged template." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259596.
Full text
4
Yao, Jianhong. "DUE-B, A NEW HUMAN DNA REPLICATION PROTEIN, IS THE FUNCTIONAL HOMOLOG OF S. CEREVISIAE SLD3." Wright State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=wright1238526547.
Full text
5
Poudel, Sumeet. "Interaction of DUE-B and Treslin during the initiation of DNA replication." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1484841026310282.
Full text
6
Chen, Xiaomi. "Aberrant DNA Replication at an Ectopic Chromosomal Site in Human Cells." Wright State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=wright1302884072.
Full text
7
Xie, Dan, and 謝丹. "Application of high-throughput tissue microarray technology in cancer research." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30283619.
Full text
8
Serrano, Moises A. "Novel Roles of Replication Protein A Phosphorylation in Cellular Response to DNA Damage." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etd/1206.
Full textAbstract:
Human replication protein A (RPA) is an eukaryotic single-stranded DNA binding protein directly involved in a variety of DNA metabolic pathways including replication, recombination, DNA damage checkpoints and signaling, as well as all DNA repair pathways. This project presents 2 novel roles of RPA in the cellular response to DNA damage. The first elucidates the regulation of RPA and p53 interaction by DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) in homologous recombination (HR). HR and nonhomologous end joining (NHEJ) are 2 distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-PK, the core component of NHEJ, partners with DNA-damage checkpoint kinases ATM, and ATR to synergistically regulate HR repair of DSBs. The regulation was accomplished through modulation of the p53-RPA interaction. We show that upon DNA damage p53 and RPA are freed from the p53–RPA complex. This is done through simultaneous phosphorylation of RPA by DNA-PK, and p53 by ATR and ATM. Neither the phosphorylation of RPA nor that of p53 alone could dissociate the p53-RPA complex; furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR and NHEJ repair through the coregulation of p53–RPA interaction by DNA-PK, ATM and ATR.
The second part of this project reveals a novel role of RPA32 phosphorylation in suppressing the signaling of programmed cell death, also known as apoptosis. Our results show that deficiency in RPA32 phosphorylation leads to increased apoptosis after genotoxic stress. Specifically, PARP-1 cleavage, Caspase-3 activation, sub-G1 cell population, annexin V staining and the loss of mitochondrial membrane potential were significantly increased in the phospho-deficient RPA32 cells (PD-RPA32). The lack of RPA phosphorylation also promoted activation of initiator Caspase-9 and effector Caspase-3 and -7. This regulation is dependent on the kinase activity of DNA-PK and is mediated by PUMA through the ATM-p53 pathway. Our results suggest a novel role of RPA phosphorylation in apoptosis that illuminates a new target that lies on the crossroads of DNA repair and cell death, a pivotal point that could be of importance for sensitizing cancer cells to chemotherapy.
9
Tower, Dallas Lauren. "The effect of putative vesicular stomatitis virus methyltransferase mutants on transcription and replication." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0010088.
Full textAbstract:
Thesis (M.S.)--University of Florida, 2005.Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.
10
Strahl, Audra Lynne. "Characterization of temperature sensitive vaccinia virus mutants in the a3l and e6r complementation groups." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0005203.
Full textAbstract:
Thesis (M.S.)--University of Florida, 2004.Typescript. Title from title page of source document. Document formatted into pages; contains 59 pages. Includes Vita. Includes bibliographical references.
11
Jeiranian, Harout Arthur. "Use of Two-Dimensional Agarose-Gel Analysis to Characterize Processing of UV-Irradiated Plasmids and the Composition of the Replisome Following UV-induced Arrest." PDXScholar, 2012. https://pdxscholar.library.pdx.edu/open_access_etds/921.
Full textAbstract:
In this thesis, I address two fundamental questions related to our understanding of how DNA damage is processed and repaired during replication. Using Two-dimensional (2-D) agarose gel analysis, I first examine whether DNA damage on plasmids introduced by transformation is processed in a manner similar to that observed on endogenously replicating plasmids and the chromosome. The original intent for using this approach was to develop a technique that could examine how different DNA adducts would be repaired in various sequence contexts. However, I found that distinct differences exist between the processing of DNA damage on transforming plasmids and the chromosome. The 2-D agarose gel analysis shows that RecA-mediated processing does not contribute to the survival of transforming plasmids and that this effect is likely due to inefficient replication of the plasmids after they are initially introduced into cells. These observations, while important, place limitations on the usefulness of transforming plasmids to characterize cellular repair processes. In a second question, I characterize the composition of the replisome following arrest by UV-induced DNA damage. Using 2-D agarose gel analysis the structural changes that occur in DNA during processing and repair have been well characterized, however, little is known about the fate of the replisome itself during these events. I used thermosensitive replication mutants to compare the DNA structural intermediates induced after disruption of specific components of the replisome to those observed after UV damage. The results show that dissociation of subunits required for polymerase stabilization are sufficient to induce the same processing events observed after UV damage. By contrast, disruption of the helicase-primase complex induces abnormal structures and a loss of replication integrity, suggesting that these components remain intact and bound to the template following replication arrest. I propose that polymerase dissociation provides a mechanism that allows repair proteins to gain access to the lesion while retention of the helicase serves to maintain the integrity and licensing of the fork so that replication can resume from the appropriate site once the lesion has been processed.
12
Latusek, Robert. "The role of LATS1 in DNA damage signalling." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:7f21ecaf-20a6-45a9-8403-c8255955920e.
Full textAbstract:
Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.
13
Perl, Abbey Leigh. "Leveraging Small Molecule Activators of Protein Phosphatase 2A (PP2A) toElucidate PP2As Role in Regulating DNA Replication and Apoptosis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1574418174603893.
Full text
14
Borgiel, Björn. "En studie för att kontrollera känsligheten av primers för lake (Lota lota), lax (Salmo salar) och öring (Salmo trutta)." Thesis, Karlstads universitet, Institutionen för miljö- och livsvetenskaper (from 2013), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-67611.
Full textAbstract:
eDNA is a fast and popular method to collect information about species presence in the environment. eDNA is DNA that is collected from environmental samples, such as water, from DNA that is expelled from organisms interacting with their environment. eDNA is an effective way to find species with small populations and alien species. There are two ways to analyze eDNA, with high-throughput DNA sequencing methods and DNA metabarcoding or use of species-specific primers and PCR. In this study, we focus on the latter, designing species-specific primers for Burbot, Brown trout, and Atlantic Salmon, testing their validity in detecting eDNA of these species with functional PCR. We also evaluated eDNA collection methods, testing different scenarios in aquarium tanks with different number of dead and alive fishes. Primers and experimental setup such as use of different temperatures of the PCR reaction used in this study didn’t result in a functional PCR as determined by electrophoresis gel. There are some problems with the design of the PCR methods for eDNA since the purpose is to design methods that can identify certain species. However, future development of eDNA methods will probably include sequencing and not detection of PCR product sizes. eDNA methods will complete traditional trapping methods like net and electrofishing, but not replace them.eDNA är en snabb och populär metod för att samla information om arters abundans i miljön. eDNA är DNA taget från miljö prover, så som vatten, från DNA som frigörs från organismer som intrigerar med deras miljön. eDNA är ett effektivt sätt att hitta arter med små populationer och främmande arter. De finns två olika sätt att analysera eDNA, med DNA-sekvenseringsmetoder med hög genomströmning och DNA-metabarkodning eller användning av artspecifika primrar och PCR. I denna studie fokuserade vi på den senare, designade artspecifika primers för lake, lax och öring, testa deras validitet vid detektering av eDNA hos dessa arter med funktionell PCR. Vi utvärderade också eDNA-insamlingsmetoder, testa olika scenarier i akvarietankar med olika antal döda och levande fiskar. Primers och experimentell uppställning, såsom användning av olika temperaturer för PCR-reaktionen som användes i denna studie, resulterade inte i en funktionell PCR såsom bestämd av elektroforesgel. Det finns några problem med utformningen av PCR-metoderna för eDNA, eftersom syftet är att utforma metoder som kan identifiera vissa arter. Emellertid kommer framtida utveckling av eDNA-metoder troligtvis att inkludera sekvensering och inte detektering av PCR-produktstorlekar. eDNA-metoder kommer att komplettera traditionella fångstmetoder som nät- och elektrofiske, men inte ersätta dem.
15
Motea, Edward A. "Probing the Chemistry and Enzymology of Translesion DNA Synthesis: Applications in Developing a Novel “Theranostic” Agent against Leukemia." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1323382718.
Full text
16
Mishra, Akaash K. "Developing small molecule inhibitors targeting Replication Protein A for platinum-based combination therapy." Thesis, 2014. http://hdl.handle.net/1805/6466.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.
17
Deem, Angela Kay. "Genome-destabilizing and Mutagenic Effects of Break-induced Replication in Saccharomyces cerevisiae." 2011. http://hdl.handle.net/1805/2625.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.
18
Vasan, Soumini. "Cascades of genetic instability resulting from compromised break-induced replication." Thesis, 2013. http://hdl.handle.net/1805/5047.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.
19
Ayyar, Sandeep. "The Molecular Mechanism of Break Induced Replication." 2013. http://hdl.handle.net/1805/3225.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.
20
"Bootstrap method to replicability: a nonparametric approach to Killeen's (2005) Prep." 2014. http://repository.lib.cuhk.edu.hk/en/item/cuhk-1291267.
Full textAbstract:
Killeen's (2005) Prep is an estimator of the replicability of an experiment. It is specifically defined as the probability of obtaining an effect of the same sign as that found in original experiment. Nevertheless, since it was announced, the validity and reliabliltiy of Prep has been challenged by a number of researchers. The present study aims at improving the performance of Prep by applying the nonparametric bootstrap method in its computation, and this bootstrap replication estimator is denoted as PBrep . A simulation study was carried out to compare the performance of Killeen's Prep and the proposed PBrep under different conditions. As expected, PBrep gives a more accurate estimation than Prep. However, PBrep occasionally fails to work properly when there is a zero population effect size, so there is still a room for improvement.Killeen (2005) 發明的Prep是一種實驗重複估計量，它是指能夠獲得與最初實驗效應量一致方向的可能性。但自其發表以來，該系數的信度及效度仍受到不少學者的質疑。是次研究嘗試通過使用自助抽樣法以改善此系數的效能，並將改良的新系數命名為PBrep。不同環境下對兩個系數準確度的模擬測試結果顯示，PBrep比Prep能達到更準確的估計值。然而當目標總體不存在差別效應時，PBrep偶爾會出現較大的偏差，因此未來研究仍需在此方向作出改善。
Chan, Man Lok.
Thesis M.Phil. Chinese University of Hong Kong 2014.
Includes bibliographical references (leaves 37-40).
Title from PDF title page (viewed on 14, September, 2016).
21
Malik, Raleigh Elizabeth. "The role of DNA methylation in regulating LHX3 gene expression." Thesis, 2014. http://hdl.handle.net/1805/4039.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.
22
Beach, Lisa Renae. "Evaluation of storage conditions on DNA used for forensic STR analysis." Thesis, 2014. http://hdl.handle.net/1805/5676.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.
23
Tumia, Rima Ahmed N. Hashm. "Role of eIF3a expression in cellular sensitivity to ionizing radiation treatments by regulating synthesis of NHEJ repair proteins." Thesis, 2015. http://hdl.handle.net/1805/9767.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Translation Initiation in protein synthesis is a crucial step controlling gene expression that enhanced by eukaryotic translation initiation factors (eIFs). eIF3a, the largest subunit of eIF3 complexes, has been shown to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of proteins important for repair of double strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a up-regulation sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) activity by suppressing expression level of NHEJ repair proteins such as DNA-PKcs and vice versa. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies including ionizing radiation.
24
Woods, Derek S. "The influence of the Ku80 carboxy-terminus on activation of the DNA-dependent protein kinase and DNA repair is dependent on the structure of DNA cofactors." Thesis, 2014. http://hdl.handle.net/1805/4665.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.
25
Desai, Akshay A. "Data analysis and creation of epigenetics database." Thesis, 2014. http://hdl.handle.net/1805/4452.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)This thesis is aimed at creating a pipeline for analyzing DNA methylation epigenetics data and creating a data model structured well enough to store the analysis results of the pipeline. In addition to storing the results, the model is also designed to hold information which will help researchers to decipher a meaningful epigenetics sense from the results made available. Current major epigenetics resources such as PubMeth, MethyCancer, MethDB and NCBI’s Epigenomics database fail to provide holistic view of epigenetics. They provide datasets produced from different analysis techniques which raises an important issue of data integration. The resources also fail to include numerous factors defining the epigenetic nature of a gene. Some of the resources are also struggling to keep the data stored in their databases up-to-date. This has diminished their validity and coverage of epigenetics data. In this thesis we have tackled a major branch of epigenetics: DNA methylation. As a case study to prove the effectiveness of our pipeline, we have used stage-wise DNA methylation and expression raw data for Lung adenocarcinoma (LUAD) from TCGA data repository. The pipeline helped us to identify progressive methylation patterns across different stages of LUAD. It also identified some key targets which have a potential for being a drug target. Along with the results from methylation data analysis pipeline we combined data from various online data reserves such as KEGG database, GO database, UCSC database and BioGRID database which helped us to overcome the shortcomings of existing data collections and present a resource as complete solution for studying DNA methylation epigenetics data.
26
Carter, Megan Elizabeth. "Blood on FTA™ Paper: Does Punch Location Affect the Quality of a Forensic DNA Profile?" 2013. http://hdl.handle.net/1805/3244.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.
27
Wang, Jiachen. "Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylation." Thesis, 2014. http://hdl.handle.net/1805/4211.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.
28
Huang, Wei. "Small molecule compounds targeting DNA binding domain of STAT3 for inhibition of tumor growth and metastasis." Thesis, 2014. http://hdl.handle.net/1805/5221.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including proliferation, survival, self-renewal, migration, invasion, angiogenesis and immune response by regulating the expression of diverse downstream target genes. Thus, inhibiting STAT3 promises to be an attractive strategy for treatment of advanced tumors with metastatic potential. We firstly identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding site of STAT3 using an in-silico screening approach; however, inS3-54 was finally found not to be appropriate for further studies because of low specificity on STAT3 and poor absorption in mice. To develop an effective and specific STAT3 inhibitor, we identified 89 analogues for the structure-activity relationship analysis. By using hematopoietic progenitor cells isolated from wild-type and STAT3 conditional knockout mice, further studies showed that three analogues (A18, A26 and A69) only inhibited STAT3-dependent colony formation of hematopoietic progenitor cells, indicating a higher selectivity for STAT3 than their parental compound, inS3-54. These compounds were found to (1) inhibit STAT3-specific DNA binding activity; (2) bind to STAT3 protein; (3) suppress proliferation of cancer cells harboring aberrant STAT3 signaling; (4) inhibit migration and invasion of cancer cells and (5) inhibit STAT3-dependent expression of downstream targets by blocking the binding of STAT3 to the promoter regions of responsive genes in cells. In addition, A18 can reduce tumor growth in a mouse xenograft model of lung cancer with little effect on body weight. Taken together, we conclude that it is feasible to inhibit STAT3 by targeting its DNA-binding domain for discovery of anticancer therapeutics.
29
Kumar, Sudhanshu. "Identification and characterization of Ascl1-expressing cells in maternal liver during pregnancy." Thesis, 2014. http://hdl.handle.net/1805/4844.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)During pregnancy, maternal liver exhibits robust growth to meet the metabolic demands of the developing placenta and fetus. Although hepatocyte hypertrophy and hyperplasia are seen in the maternal liver, the molecular and cellular mechanisms mediating the maternal hepatic adaptations to pregnancy is poorly understood. Previous microarray analysis revealed a most upregulated gene named Ascl1, a transcription factor essential for neural development, in the maternal liver at mid-gestation. The aims of the study were to (1) validate the activation of Ascl1 gene; (2) identify Ascl1-expressing cells; and (3) determine the fate of Ascl1-expressing cells, in the maternal liver during the course of gestation. Timed pregnancy was setup in mice and the maternal livers were collected at various stages of gestation. Maternal hepatic Ascl1 mRNA expression was evaluated by qRT-PCR and northern blotting. The results demonstrated that the transcript level of maternal hepatic Ascl1 is exponentially increased during the second half of pregnancy in comparison with a non-pregnant state. Using a Ascl1-GFP mouse model generated by others to monitor the behavior of neural progenitor cells, we found that maternal hepatic Ascl1-expressing cells are non-parenchymal cells, very small in size, and expanding during pregnancy. To map the fate of this cell population, we generated an in vivo tracing mouse model named Ascl1-CreERT2/ROSA26-LacZ. Using this model, we permanently labeled maternal hepatic Ascl1-expressing cells at midgestation by giving tamoxifen and analyzed the labeled cells in the maternal liver prior to parturition. We observed that the initial small Ascl1-expressing cells undergoing expansion at mid-gestation eventually became hepatocyte-like cells at the end stage of pregnancy. Taken together, our findings strongly suggest that Ascl1-expressing cells represent a novel population of hepatic progenitor cells and they can differentiate along hepatocyte lineage and contribute to pregnancy-induced maternal liver growth. Further studies are needed to firmly establish the nature and property of maternal hepatic Ascl1-expressing cells. At this stage, we have gained significant insights into the cellular mechanism by which the maternal liver adapts to pregnancy.
30
Rainey, Christina. "Identification of Tobacco-Related Compounds in Tobacco Products and Human Hair." Thesis, 2014. http://hdl.handle.net/1805/4979.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Analyses of tobacco products and their usage are well-researched and have implications in analytical chemistry, forensic science, toxicology, and medicine. As such, analytical methods must be developed to extract compounds of interest from tobacco products and biological specimens in order to determine tobacco exposure. In 2009, R.J. Reynolds Tobacco Co. released a line of dissolvable tobacco products that are marketed as a smoking alternative. The dissolvables were extracted and prepared by ultrasonic extractions, derivatization, and headspace solid phase microextraction (SPME) with analysis by gas chromatography-mass spectrometry (GC-MS). The results show that the compounds present are nicotine, flavoring compounds, humectants and binders. Humectant concentrations vary among different tobacco types depending on the intended use. Humectants were quantified in various tobacco types by GC and “splitting” the column flow between a flame ionization detector (FID) and an MS using a microfluidic splitter in order to gain advantage from the MS’s selectivity. The results demonstrated excellent correlation between FID and MS and show that MS provides a higher level of selectivity and ensures peak purity. Chemometrics was also used to distinguish products by tobacco type. Hair is a common type of evidence in forensic investigations, and it is often subjected to mitochondrial DNA (mtDNA) analysis. Preliminary data was gathered on potential “lifestyle” markers for smoking status as well as any indications of subject age, gender, or race by investigating the organic “waste” produced during a mtDNA extraction procedure. The normally discarded organic fractions were analyzed by GC-MS and various lipids and fatty acids were detected. At this point, a total vaporization-SPME (TV-SPME) method was theorized, developed, and optimized for the specific determination of nicotine and its metabolite, cotinine. The theory of TV-SPME is to completely vaporize an organic extract which will eliminate the partitioning between the sample and the headspace, thereby simplifying the thermodynamic equilibrium. Parameters such as sample volume, incubation temperature, and extraction time were optimized to achieve the maximum analyte signal. Response surface methodology (RSM) is a statistical model that is very useful in predicting and determining optimum values for variables to ensure the ideal response. RSM was used to optimize the technique of TV-SPME for the analysis of nicotine and cotinine. Lastly, quantitation of nicotine and cotinine in human hair typically requires large sample sizes and extensive extraction procedures. Hence, a method using small sample sizes and a simple alkaline digestion followed by TV-SPME-GC-MS has been developed. Hair samples were collected from anonymous volunteers and nicotine and cotinine were identified and quantitated in the hair of tobacco users.
31
Pham, Duy. "Twist1 and Etv5 are part of a transcription factor network defining T helper cell identity." Thesis, 2014. http://hdl.handle.net/1805/4657.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)CD4 T helper cells control immunity to pathogens and the development of inflammatory disease by acquiring the ability to secrete effector cytokines. Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu by activating Signal Transducer and Activator of Transcription factors that induce the expression of other transcription factors important for cytokine production. STAT4 is a critical regulator of Th1 differentiation and inflammatory disease that attenuates the gene-repressing activity of Dnmt3a. In the absence of STAT4, genetic loss of Dnmt3a results in de-repression of a subset of Th1 genes, and a partial increase in expression that is sufficient to observe a modest recovery of STAT4-dependent inflammatory disease. STAT4 also induces expression of the transcription factors Twist1 and Etv5. We demonstrate that Twist1 negatively regulates Th1 cell differentiation through several mechanisms including physical interaction with Runx3 and impairing STAT4 activation. Following induction by STAT3-activating cytokines including IL-6, Twist1 represses Th17 and Tfh differentiation by directly binding to, and suppressing expression of, the Il6ra locus, subsequently reducing STAT3 activation. In contrast, Etv5 contributes only modestly to Th1 development but promotes Th differentiation by directly activating cytokine production in Th9 and Th17 cells, and Bcl6 expression in Tfh cells. Thus, the transcription factors Twist1 and Etv5 provide unique regulation of T helper cell identity, ultimately impacting the development of cell-mediated and humoral immunity.
32
Chen, Yuanyuan. "Epigenetic alteration by prenatal alcohol exposure in developing mouse hippocampus and cortex." Thesis, 2014. http://hdl.handle.net/1805/5810.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.
33
Andere, Anne A. "De novo genome assembly of the blow fly Phormia regina (Diptera: Calliphoridae)." Thesis, 2014. http://hdl.handle.net/1805/5630.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Phormia regina (Meigen), commonly known as the black blow fly is a dipteran that belongs to the family Calliphoridae. Calliphorids play an important role in various research fields including ecology, medical studies, veterinary and forensic sciences. P. regina, a non-model organism, is one of the most common forensically relevant insects in North America and is typically used to assist in estimating postmortem intervals (PMI). To better understand the roles P. regina plays in the numerous research fields, we re-constructed its genome using next generation sequencing technologies. The focus was on generating a reference genome through de novo assembly of high-throughput short read sequences. Following assembly, genetic markers were identified in the form of microsatellites and single nucleotide polymorphisms (SNPs) to aid in future population genetic surveys of P. regina. A total 530 million 100 bp paired-end reads were obtained from five pooled male and female P. regina flies using the Illumina HiSeq2000 sequencing platform. A 524 Mbp draft genome was assembled using both sexes with 11,037 predicted genes. The draft reference genome assembled from this study provides an important resource for investigating the genetic diversity that exists between and among blow fly species; and empowers the understanding of their genetic basis in terms of adaptations, population structure and evolution. The genomic tools will facilitate the analysis of genome-wide studies using modern genomic techniques to boost a refined understanding of the evolutionary processes underlying genomic evolution between blow flies and other insect species.