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Philosophiae Doctor - PhD / 2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.

Polypurine·polypyridine (pur·pyr) tracts are a run of all purines on one strand and all pyrimidines on the complementary DNA strand. Statistical overrepresentation of the tracts in eukarocytes suggests a cellular role or roles. The tracts from triplex DNA <i>in vitro</i> and there is evidence for triplex DNA <i>in vivo</i>. Several cellular roles are possible for triplex DNA. The presence of the tracts in gene 5' flanking regions suggets a regulatory role. This work investigates the role of triplex DNA in the cell, particularly in the regulation of transcription. Proteins mediate DNA looping in the regulation of transcription and in its condensation in chromosomes. Such looping may also be mediated by transmolecular triplexes, formed between separated pur·pyr tracts. Formation of pyr·pur·pyr transmolecular triplexes was investigated using linear and circular plasmid models containing separated pur·pyr tracts able to form a triplex with each other, but not within a tract. Transmolecular triplex loops (T-loops) formed in circular DNA, suggesting a possible regulatory or structural role <i>in vivo</i>. The following model shows a T-loop formed at pH 4. At pH 6, a duplex partially reforms and single-stranded region(s) trap the structure. and single-stranded region(s) trap the structure. T-loops were used as a model to test the Idea that a single-strand extruded by triplex formation in the 5' flanking region of a gene could promote transcription. Transcription was inhibited in T-loops, suggesting such structures could block transcriptional elongation if formed <i>in vivo</i>. The ability of polyamine analogues to promote triplex formation was also tested using T-loops. Pentamines promoted T-loop formation at lower concentrations than tetramines. Spatial distribution of charge was also important. A triplex role in transcriptional regulation was investigated using two examples of human genes with 5' flanking pur·pyr tracts. The effect of triplex-specific antibodies on expression of c-' myc' was investigated using agarose-encapsulated nuclei. Triplex formation between c-'src' promoter pur·pyr tracts was visualized as gel band shift die to dimerization between linear plasmid fragments containing individual tracts. A transmolecular triplex was proposed as one way in which the c-'src' tracts could form a triplex <i>in vivo</i> which might be involved in the regulation of transcription.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Filter cake and Triplex are powdered by-products of aluminum sulfate and soap factories, respectively. Studies were designed to determine elemental composition of these two powders and evaluate the efficacy against stored product insect species on concrete surfaces and commodities. Elemental composition of the powders was determined using conjugated scanning electron microscopy and Energy-dispersive X-ray spectroscopy. No heavy metals were found in both powders, and the dominant elements found were silicon and oxygen in the form of silicon dioxide. The efficacy of filter cake and Triplex against the maize weevil, Sitophilus zeamais Motschulsky; rice weevil, Sitophilus oryzae (Linnaeus); lesser grain borer, Rhyzopertha dominica Fabricius; red flour beetle, Tribolium castaneum (Herbst); saw-toothed grain beetle, Oryzaephilus surinamensis (Linnaeus); and Indian meal moth, Plodia interpunctella (Hübner), was determined using a range of concentrations and exposure times. On concrete surfaces ≥ 7.5 g/m² of filter cake produced more than 99% mortality of S. zeamais and S. oryzae adults within 12–24 h, whereas more than three times the concentration of filter cake was required to achieve similar mortality of both species in Triplex treatments. At 3 g/m² of filter cake, 99% mortality S. zeamais and S. oryzae adults was achieved within 22–27 h of exposure. The corresponding exposure time at 9 g/m² of Triplex was 39 h to achieve 99% mortality of both species. For both powders, lower concentrations and exposure times were required to achieve complete suppression of progeny production, percentage of insect damaged kernels, and percentage of grain weight loss compared to the concentrations and exposure times required for 00% mortality. Filter cake treated wheat at concentrations above 0.7 g/kg produced more than 99% mortality of S. zeamais and S. oryzae adults. Similarly, filter cake concentrations above 2 g/kg on wheat produced more than 99% mortality of R. dominica, T. castaneum, and O. surinamensis adults. However, on maize ≥ 3 g/kg of filter cake concentration was required to achieve similar mortality of R. dominica, T. castaneum, and O. surinamensis. Higher concentrations of Triplex were required to achieve similar mortalities of tested species on maize and wheat. Reduction in progeny production was greater when adults were exposed to higher concentrations than lower concentrations. Complete suppression of live larvae and adult emergence of P. interpunctella was achieved after exposure of eggs for 21 and 42 d to ≥ 2 g/kg of filter cake treated maize and to ≥ 0.5 g/kg of filter cake treated wheat. Similarly, complete suppression of live larvae and adult emergence was achieved when eggs were exposed to ≥ 6 g/kg of Triplex treated maize and to 3g/kg of Triplex treated wheat. In general, our study consistently showed that filter cake was more efficacious compared to Triplex against all tested species on both surfaces and commodities. Filter cake and Triplex should be recommended for protecting grain stored by smallholder farmers in Ethiopia to discourage farmers from using dangerous chemical insecticides. However, field studies should be done using both powders against stored product insects in smallholder farmers’ traditional storages structures in Ethiopia to determine concentrations that are practical under field conditions. The effective duration of protection offered by these powders should also be investigated.

Triplex forms of DNA act as main factors of some important cell functions. However, their positions within genome and their effect on cell functions are not known well. Triplex search algorithms often don't consider many of triplexs features and the possibility of occurrence of errors. In the other hand the complexity of full featured algorithms is extremely high. This paper shows the way to speed up the algorithm that considers all known triplex features. Parallel aproach allows due to CUDA technology acceleration up to 50.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19527号 / 理博第4187号 / 新制||理||1601(附属図書館) / 32563 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

DNA translocases are molecular motors that use energy from nucleotide triphosphate (NTP) hydrolysis to move along, pump, remodel or clear DNA. Unlike helicases, double-stranded DNA (dsDNA) translocases do not unwind DNA; their action has no net product apart from inducing supercoils as a result of groove-tracking, which has hampered their characterisation. Many dsDNA translocases appear to have biased directionality. However, the inherent symmetry of dsDNA requires that translocase activity is regulated by specific sequences or through modulation by interaction partners. FtsK is a highly conserved bacterial cell-division protein, localised to the dividing septum, that coordinates chromosome segregation with cytokinesis. It is responsible for the resolution of chromosome dimers by activating the tyrosine recombinases XerCD bound to the 28bp chromosomal site dif. The C-terminal domain of FtsK (FtsKC) is a dsDNA translocase (speed ~5 kb/s, stall force ~60 pN) most closely related to superfamily 4 helicases and is active as a hexameric ring. A winged-helix subdomain at the C-terminus of FtsKC, FtsKgamma, binds to specific 8 bp sequences, KOPS, that are polarised in the bacterial chromosome from the origin to towards dif. FtsKgamma also interacts with XerD, activating it for catalysis. Studies of FtsK translocation have differed over whether KOPS act as a loading or a reversal sequence for FtsK. In Chapter 2, I use a continuous ensemble assay for dsDNA translocation to show that FtsK initiates rapidly at KOPS, with loading dependent on FtsKgamma. Translocation requires moderately cooperative ATP binding, while ATP hydrolysis has a more relaxed cooperativity. I have determined the ATP coupling efficiency of translocation to be ~1.6 bp/ATP, in line with theoretical estimates. Though FtsK probably strips most proteins from DNA, I show in Chapter 3 that FtsK stops translocating when it encounters XerCD bound to dif. The interaction is most likely a specific down-regulation, but surprisingly does not depend on FtsKgamma or on the catalytic or synaptic activity of XerCD. In Chapter 4, I show some preliminary structural data of FtsKC bound to dsDNA, with the aim of determining the first high resolution structure of a ring dsDNA translocase bound to nucleic acid.

Thesis advisor: Larry W. McLaughlin / The ability to control gene expression has traditionally been pursued at the protein level, using drugs designed to mimic a natural substrate or to disrupt a protein's active site. Traditional drug targeting by competitive and non-competitive inhibitors, however, requires a fairly detailed knowledge of the target protein's three-dimensional structure. More recently, focus has broadened to include alternative methods of genetic control, including the use of single-stranded DNA or RNA probe sequences which control gene expression by targeting the genes themselves. Within the last two decades, peptide nucleic acids (PNAs) – DNA mimics possessing natural bases linked to an N-(2-aminoethyl)-glycine (AEG) backbone – have proven as effective in gene-targeting as traditional synthetic DNA or RNA with the added advantages of tighter binding and greater specificity. Additionally, PNAs are not easily recognized by nucleases, proteases, and peptidases giving them greater resistance to enzyme degradation and making them even more favorable for gene targeting in vivo. Traditional PNA triplexes are composed of two polypyrmidine PNA strands bound to the Watson-Crick and Hoogsteen faces, respectively, of the polypurine strand of target DNA after displacing the polypyrimidine strand of the original DNA duplex. Janus Wedge (JW) residues, on the other hand, utilize unnatural bases linked to the AEG backbone, which are capable of hydrogen bonding to the Watson-Crick faces of both strands of a target DNA duplex. JW triplex formation, then, has a DNA2-PNA stoichiometry, and no Hoogsteen face interactions. The generalization of the DNA duplex targeting strategy by peptide oligomers requires substantial discoveries in the field of PNA research, including an understanding of the three-dimensional structure and folding pattern of these triple-stranded molecules. This report details the crystallization efforts on JW DNA-peptide-DNA triplexes using 11dC811-11T811 target sequences – with and without single base overhangs – and synthetic W8K peptide. Hanging drop vapor diffusion methods showed that while crystal formation was extremely elusive, in narrowing the optimal buffer conditions, 25% PEG concentration was consistently correlated with the most promising crystallization efforts for both the overhanged and non-overhanged sequences. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.