Thesis (PH. D.)--Michigan State University. Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 103-112).

Thesis (Ph. D.)--School of Dentistry and School of Pharmacy. University of Missouri--Kansas City, 2004. / "A dissertation in oral biology and pharmacology." Advisor: J. David Eick. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 22, 2006. Includes bibliographical references (leaves 176-189). Online version of the print edition.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 69-72).

O presente trabalho apresenta resultados de preparação, caracterização e aplicação de membranas de DNA e DNA-PEDOT:PSS como material transportador de buracos (HTM) em células solares sensibilizadas por corante (DSSC). As análises por UV-Vis das amostras revelaram 80% de transparência em 600 nm para o DNA-isolado e diminuindo para 62% em 550 nm para o DNA-2% PEDOT:PSS. As análises das amostras por FTIR revelaram os picos característicos tanto do DNA quanto do PEDOT:PSS, confirmando a incorporação deste último nas membranas. Os resultados de análises térmicas DSC evidenciaram a presença de Tg em -67ºC e o seu desaparecimento com a adição de PEDOT:PSS na formulação de HTM. As análises de TGA mostraram o aumento da estabilidade das amostras com a adição de PEDOT:PSS atingindo 200ºC. Todas as amostras apresentaram 19% de resíduos em 900ºC. As membranas a base de DNA também foram submetidas às medidas de condutividade iônica revelando o maior valor de 3,2x10-4 S/cm2 em temperatura ambiente e aumentando para 0,1x10-3 S/cm2 em temperatura de 75ºC, para a amostra de DNA-isolado. As amostras de DNA-PEDOT:PSS mostraram valores de condutividade de 4,67x10-5 S/cm2 para a amostra DNA -2% PEDOT:PSS, em temperatura ambiente e diminuíram com o aumento de porcentagem de PEDOT:PSS. Dos difratogramas de raios-X observou-se um aumento da cristalinidade das amostras com a adição de PEDOT:PSS sendo o maior valor encontrado de 77,8% foi para a amostra de DNA-10% PEDOT:PSS. As DSSCs apresentaram a diminuição de eficiência solar após a introdução de membranas de DNA de 2.04% para 1,49% fenômeno explicado em termos de aumento de reflectância e rugosidade das amostras que dificultou o transporte de carga e recombinação do par redox do eletrólito nas células solares sensibilizadas por corante. / This paper presents results of preparation, characterization and application of DNA and DNA-PEDOT:PSS-based membranes as hole-carrier material (HTM) in dye-sensitized solar cells (DSSC). The UV-Vis analysis of the samples revealed 80% of transparency at 600 nm for the isolated DNA and 62% at 550 nm for DNA-2% PEDOT:PSS. The FTIR analysis of the samples showed characteristic peaks of both the DNA and PEDOT:PSS, confirming its incorporation into membranes. The results of DSC analysis revealed the presence of Tg at -67ºC and its disappearance with the addition of PEDOT:PSS to the formulation of HTM. The TGA analysis showed increased stability of the samples with the addition of PEDOT:PSS reaching 200ºC. All samples showed 19% of ashes at 900ºC. The DNA-based membranes were also subjected to ionic conductivity measurements showing the highest value of 3.2x10-4 S/cm2 at room temperature and of 0.1x10-3 S/cm2 at 75ºC for the isolated DNA. Samples of DNA-PEDOT:PSS showed conductivity value of 4.67x10-5 S/cm2 for DNA-2% PEDOT:PSS sample at room temperature which decreased with increasing percentage of PEDOT:PSS. X-ray diffraction revealed an increase of the crystallinity of the samples with the addition of PEDOT:PSS and the highest value found was 77.8% for the sample of DNA-10% PEDOT:PSS. The DSSCs showed a reduction of solar efficiency from 2.04% to 1.49% after the introduction of DNA-based membranes. This phenomenon was explained in terms of increased reflectance and surface roughness of the samples that difficult the transport and recombination of charge carrier species.

abstract: DNA is a unique, highly programmable and addressable biomolecule. Due to its reliable and predictable base recognition behavior, uniform structural properties, and extraordinary stability, DNA molecules are desirable substrates for biological computation and nanotechnology. The field of DNA computation has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. This dissertation focuses on building novel types of computational DNA systems based on both DNA reaction networks and DNA nanotechnology. A series of related research projects are presented here. First, a novel, three-input majority logic gate based on DNA strand displacement reactions was constructed. Here, the three inputs in the majority gate have equal priority, and the output will be true if any two of the inputs are true. We subsequently designed and realized a complex, 5-input majority logic gate. By controlling two of the five inputs, the complex gate is capable of realizing every combination of OR and AND gates of the other 3 inputs. Next, we constructed a half adder, which is a basic arithmetic unit, from DNA strand operated XOR and AND gates. The aim of these two projects was to develop novel types of DNA logic gates to enrich the DNA computation toolbox, and to examine plausible ways to implement large scale DNA logic circuits. The third project utilized a two dimensional DNA origami frame shaped structure with a hollow interior where DNA hybridization seeds were selectively positioned to control the assembly of small DNA tile building blocks. The small DNA tiles were directed to fill the hollow interior of the DNA origami frame, guided through sticky end interactions at prescribed positions. This research shed light on the fundamental behavior of DNA based self-assembling systems, and provided the information necessary to build programmed nanodisplays based on the self-assembly of DNA. / Dissertation/Thesis / Ph.D. Chemistry 2014

Indiana University-Purdue University Indianapolis (IUPUI) / Modern DNA profiling techniques have increased in sensitivity allowing for higher success in producing a DNA profile from limited evidence sources. However, this can lead to the amplification of more DNA profiles that do not get a hit on a suspect or DNA database and more mixture profiles. The work here aims to address or improve these consequences of current DNA profiling techniques. Based on allele-specific PCR and quantitative color measurements, a 24-SNP forensic phenotypic profile (FPP) assay was designed to simultaneously predict eye color, hair color, skin color, and ancestry, with the potential for age marker incorporation. Bayesian Networks (BNs) were built for model predictions based on a U.S sample population of 200 individuals. For discrete pigmentation traits using an ancestry influenced pigmentation prediction model, AUC values were greater than 0.65 for the eye, hair, and skin color categories considered. For ancestry using an all SNPs prediction model, AUC values were greater than 0.88 for the 5 continental ancestry categories considered. Quantitative pigmentation models were also built with prediction output as RGB values; the average amount of error was approximately 7% for eye color, 12% for hair color, and 8% for skin color. A novel sequencing method, methyl-RADseq, was developed to aid in the discovery of candidate age-informative CpG sites to incorporate into the FPP assay. There were 491 candidate CpG sites found that either increased or decreased with age in three forensically relevant xii fluids with greater than 70% correlation: blood, semen, and saliva. The effects of exogenous microbial DNA on human DNA profiles were analyzed by spiking human DNA with differing amounts of microbial DNA using the Promega PowerPlex® 16 HS kit. Although there were no significant effects to human DNA quantitation, two microbial species, B. subtilis and M. smegmatis, amplified an allelic artifact that mimics a true allele (‘5’) at the TPOX locus in all samples tested, interfering with the interpretation of the human profile. Lastly, the number of contributors of theoretically generated 2-, 3-, 4-, 5-, and 6-person mixtures were evaluated via allele counting with the Promega PowerPlex® Fusion 6C system, an amplification kit with the newly expanded core STR loci. Maximum allele count in the number of contributors for 2- and 3-person mixtures was correct in 99.99% of mixtures. It was less accurate in the 4-, 5-, and 6-person mixtures at approximately 90%, 57%, and 8%, respectively. This work provides guidance in addressing some of the limitations of current DNA technologies.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.

No description available.

Translesion DNA Synthesis (TLS) is a mechanism that promotes DNA damage tolerance during DNA replication using an error-prone DNA polymerase complex. The complex is comprised of the ImuA, ImuB, and ImuC proteins that are found in approximately one-third of bacteria, including high priority antimicrobial resistant pathogens, such as Pseudomonas aeruginosa. Previous in vivo studies have shown that TLS increases beneficial bacterial mutations as the error-prone DNA polymerase, ImuC, lacks proof-reading activity. However, how ImuA and ImuB proteins contribute to the polymerase mechanism is unknown. Thus, the goal of this study is to characterize the TLS proteins in vitro to determine how ImuA and ImuB associate with ImuC to promote error- prone replication. ImuA and ImuBNΔ34 were successfully purified for biochemical characterization from the homolog Myxococcus xanthus. Using size-exclusion chromatography coupled to multi-angle light scattering, both ImuA and ImuBNΔ34 are trimers in solution. Each protein also binds DNA independently as assessed by fluorescence polarization. Interestingly, both proteins bind ssDNA and a 3’ overhang substrate mimicking the DNA replication intermediate with the highest affinity. DNA binding assays further confirm these proteins can form a DNA-ImuA-ImuBNΔ34 complex. Using bacterial two-hybrid assays, the ImuA- ImuB interaction occurs in the C-terminal region of both proteins. Overall, these results suggest that ImuA and ImuB may recruit and stabilize ImuC on DNA for replication past damaged DNA, providing the first insights into the ImuA and ImuB molecular mechanism. / Thesis / Master of Science (MSc)