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This report combines a series of papers to trace progression in the area of type-2 topoisomerase research. First, Deibler et al. show that knotted DNA is harmful to cells. Knots can block both transcription and replication, and can also act as a catalyst for mutation. Despite the fact that type-2 topoisomerases perform the important functions of unknotting and decatenating DNA, the mechanism by which they accomplish this task is still unknown. Buck and Zechiedrich propose a model in which the enzyme uses local geometry to infer global topology, and thus where to perform segment passage in order to obtain the desired results. In two articles, Liu et al. evaluate this theory quantitatively for the decatenation and unknotting problems. In both cases it is shown that the presence of certain juxtapositions is strongly correlated with global topology. This correlation is not enough, however, and Liu et al. go on to show that when segment passage operations designed to mimic type-2 topoisomerase action are performed at hooked juxtapositions, the overwhelming tendency is towards unknotting and decatenation. / text

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.

DNA condensation is a fundamental process in all living organisms. The highly abundant nucleoid-associated proteins, HU and IHF, present in bacteria, have been shown to play an important role in shaping the nucleoid. However, the exact mechanism is not well understood. In this thesis, we have demonstrated that both HU and IHF guide DNA to condense into linear bundle-like structures in presence of cellular condensing components, but the proteins alone do not condense DNA into densely packed structures. Our results suggest a mechanism by which HU and IHF could act as architectural proteins during in vitro and in vivo DNA condensation. More recently, DNA condensation has attracted much attention for its relevance in optimizing artificial DNA delivery systems for gene therapy. The research presented in this dissertation provides in depth biophysical studies that demonstrate how local modulations in the nucleic acid structure can be used to control both the size and the morphology DNA condensates. We describe a novel strategy for improving the condensation of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. kb) with flexible sites at regular intervals along the duplex backbones, in the form of single-stranded nicks or single-stranded gaps. Our results also provide new insights into the role of DNA flexibility in condensate formation and suggest the potential for the use of this DNA structure in the design of vectors for oligonucleotide and gene delivery.

DNA replication is cruciaI for the transmission of genetic information. Understanding the enzymology involved in this complex process will allow further insight into its mechanism. Experimental evidence indicates a role for DNA ligase I in DNA replication. Techniques of molecular and cellular biology and immunology were utilized in this study to further investigate DNA ligase I and clarify its involvement and interaction with other proteins in DNA replication. Immunofluorescence studies were performed to examine the intracellular distribution of DNA ligase I. Confocal analysis of indirect immunofluorescence detection of DNA ligase I using affinity purified anti-human DNA ligase I antibodies showed nuclear localization of DNA ligase I in distinct foci resembling those structures seen in detection of centres of DNA replication and other DNA replication proteins. Immunoprecipitation experiments were performed on extracts of MDBK cells to examine possible interactions of DNA ligase I with the DNA replication cofactor, PCNA; and no interactions were detected.

The technique of restriction fragment length polymorphism (RFLP) analysis, of chloroplastic and genomic DNA, was investigated as a means of identifying eucalypt species and cultivars which are morphologically indistinguishable from one another. In order to resolve chloroplast DNA (cpDNA) RFLPs, a method was developed to extract high yields of intact chloroplasts from Eucalyptus grandis S/N M6. Starch contamination was reduced by incubation of saplings in the dark for 48 h prior to extraction and watering with a solution containing 370 mM Na-phosphate and 296 mM KN03. Optimal chloroplast yields (25 ug chlorophyll/g fresh mass) were obtained by chopping leaf material, using a vertical homogenizer, in a buffer containing 350 mM sorbitol, 50 mM tris-HCL and 5 mM EDTA, 0.1 % (w/v) bovine serum albumin, 0.15 % (w/v) 2-mercaptoethanol, 2 mM L-ascorbic acid and 1 mM MgCI2 followed by washing of leaf pieces in a buffer containing only sorbitol, tris-HCL and EDTA. When these chloroplasts were used in an "in-organelle" DNA digestion procedure, polymorphisms were observed between the cpDNA profiles resolved for E. grandis S/N M6 and that of an outgroup species (spinach). However, the developed chloroplast extraction technique could not be used to obtain chloroplasts from various other eucalypt species, probably as a result of variability in the material at an ultrastructural or biochemical level. For the analysis of genomic DNA RFLPs, a DNA extraction procedure was optimized for use with various eucalypt species and cultivars. This included the development of a purifcation technique during which DNA was ammonium acetate-ethanol precipitated and subjected to mini-dialysis. Following Dra I restriction of DNA, the extract was electrophoresed and Southern blotted onto both nylon and nitrocellulose membranes. These were probed with a Hind-III restricted sample of the multilocus plasmid probe pV47-2. This probe was labelled using 32p as well as a non-radioactive labelling substance digoxygenin (DIG). Hybridization conditions, including the composition of the hybridization buffer, were optimized for use with these labels, and DNA RFLPs (fingerprints) were resolved for the eucalypt species E. grandis and E. macarthurii and cultivars of E. grandis (S/N M6, TAG 5 and TAG 14). An average of 8.5 bands were detected with 32p and 5.0 fragments with DIG. All the species and cultivars fingerprinted with the 32P-label could be distinguished from one another. However, as a result of the reduced sensitivity of the DIG system, two of the E. grandis cultivars, S/N M6 and TAG 5, could not be differentiated. It is concluded that the latter system would be most suitable for incorporation into a routine eucalypt screening programme, although it is suggested that the colourimetric detection assay, used in this study to resolve DNA bands, be replaced by a more sensitive one. / Thesis (M.Sc)-University of Natal, Durban, 1993.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Thesis (Ph.D.) - University of St Andrews, January 2007.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 125-133).