Secondary structures may have to endure severe vibration amplitudes under the influence of the primary structures on which they are mounted. A series of numerical case studies are presented in this thesis to investigate the effectiveness of a passive vibration controller which combines a conventional tuned absorber with an impact damper, to attenuate the excessive vibration amplitudes of light secondary structures. In addition, experimental measurements are reported for some selective cases and comparisons are made with numerical predictions. This suggested configuration seems to suit ideally as an add-on enhancer for existing conventional absorbers. Most of the Results are presented for random white noise excitation, and a few representative transient vibration cases are also studied. / Thesis / Master of Engineering (ME)

RNA molecules interact by forming inter-molecular base pairs that compete with the intra-molecular base pairs of their secondary structures. Here we investigate the patterns of neutral mutations in RNAs whose function is the interaction with other RNAs, i.e., the co-folding with one or more other RNA molecules. We find that (1) the degree of neutrality is much smaller in interacting RNAs compared to RNAs that just have to coform to a single externally prescribed target structure, and (2) strengthening this contraint to the conservation of the co-folded structure with two or more partners essentially eliminates neutrality. It follows that RNAs whose function depends on the formation of a specific interaction complex with a target RNA molecule will evolve much more slowly than RNAs with a function depending only on their own structure.

Unusual DNA secondary structures, especially G-quadruplexes and i-motifs, play important roles in gene transcriptional regulation and have been identified as novel drug targets. In this dissertation, I explored their formation in the human VEGF and RET promoters and their roles in gene transcriptional regulation. VEGF is a key regulator of angiogenesis and is up-regulated in many types of tumors. A poly-guanine/poly-cytosine (polyG/polyC) tract in its proximal promoter (-85 to -50 base pairs relative to the transcription starting site) is essential for both basal and inducible VEGF expression. I demonstrated that the guanine-rich (G-rich) and cytosine-rich (C-rich) strands in the VEGF proximal promoter are able to form G-quadruplex and i-motif structures, respectively. The major G-quadruplex formed by the VEGF G-rich sequence is an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:4:1 arrangement of three double-chain-reversal loops (two single-base loops and one loop with four bases). The complementary C-rich sequence in the same region forms an intramolecular i-motif containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration (two double-base loops and one loop with three bases). The Gquadruplexes formed by the native VEGF G-rich and its derivative sequences were also confirmed by NMR. In addition, various transcription factors including Sp1, hnRNP K, CNBP and nucleolin, which recognize different DNA structural elements including single-stranded, double-stranded or G-quadruplex/i-motif DNA in the VEGF proximal promoter, have been confirmed by EMSA, siRNA and chromatin immunoprecipitation (ChIP) assay, suggesting that the DNA in the VEGF proximal promoter region is capable of undergoing transitions between those three structures. Based on my studies, I have proposed a model to describe how various transcription factors recognize different DNA structures in the VEGF proximal promoter to regulate transcription. In the proximal promoter of another important oncogene RET, I demonstrated that the guanine-rich strand forms an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:3:1 arrangement of three double-chain-reversal loops. The complementary cytosine-rich strand forms an i-motif structure containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration. Moreover, G-quadruplex-interactive compounds TMPyP4 and telomestatin were shown to further stabilize the RET G-quadruplex structure.

In recent years, RNA structural comparison becomes a crucial problem in bioinformatic research. Generally, it is a popular approach for representing the RNA secondary structures with arc-annotation sets. Several methods can be used to compare two RNA structures, such as tree edit distance, longest arc-preserving common subsequence (LAPCS) and stem-based alignment. However, these methods may be helpful only for small RNA structures because of their high time complexity. In this thesis, we propose a simplified method to compare two RNA structures in O(mn)time, where m and n are the lengths of the two RNA sequences, respectively. Our method transforms the RNA structures into specific sequences called object sequences, then compare these object sequences to find their common substructures. We test our comparison method with 118 RNA structures obtained from RNase P Database. For any two structures, we try to identify whether they are in the same family by both structure comparison and sequence comparison. In our experiment, we find that our method for comparing RNA structures can yield better hit rates and is faster than the traditional method to compare the RNA sequences. Therefore, our approach for comparing RNA secondary structures is more sensitive in biology and more efficient in time complexity.

The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, the demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. We demonstrate for the first time the i-motif DNA secondary structure formed from an element within the Bcl-2 promoter region has potential to serve as a cellular molecular target for modulation of gene expression. Unlike typical oncogenes, Bcl-2 acts by promoting cellular survival rather than increasing cellular proliferation. The over-expression of Bcl-2, most notably in lymphomas, has been associated with the development of chemoresistance.Transcriptional regulation of Bcl-2 is highly complex and a guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to Bcl-2 promoter activity. We have demonstrated that the C-rich strand is capable of forming an intramolecular i-motif DNA secondary structure with a transition pH of 6.6 and a predominant 8:5:7 loop using mutational studies coupled with circular dichroic spectra and thermal stability analyses. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. Two different classes of steroids either stabilize or destabilize the i-motif structure and this differential interaction results in the activation or repression of Bcl-2 expression. The i-motif stabilizing steroid significantly up-regulated Bcl-2 gene and protein expression in BJAB Burkitt's lymphoma cells while the destabilizing steroid down-regulated Bcl-2 expression in B95.8 Burkitt's and Granta-519 mantle cell lymphoma cells, as well as in a SCID mouse lymphoma model. More importantly, the down-regulation of Bcl-2 led to chemosensitization of etoposide-resistant lymphoma cells demonstrating that Bcl-2 i-motif interactive small molecules can act as chemosensitizing agents. Conversely, compounds that up-regulate Bcl-2 by stabilization of the i-motif have potential for use as neuroprotective agents.

>Magister Scientiae - MSc / Despite widespread rubella virus (RV) vaccination programs, annually RV still causes severe congenital defects in an estimated 100,000 children globally. A concerted attempt to eradicate RV is currently underway and analytical tools to monitor the global decline of the last remaining RV lineages will be useful for assessing the effectiveness of this endeavour. Importantly, RV evolves rapidly enough that much of its epidemiological information might be inferable from RV genomic sequence data. Using BEASTv1.8.0, I analysed publically available RV sequence data to estimate genome-wide and gene-specific nucleotide substitution rates, to test whether the current estimates of RV substitution rates are representative of the entire RV genome. During these investigations, I specifically accounted for possible confounders of nucleotide substitution rate estimates, such as temporally biased sampling, sporadic recombination, and natural selection favouring either increased or decreased genetic diversity (estimated by the PARRIS and FUBAR methods) at nucleotide sites within RV nucleic acid secondary structures (predicted by the NASP method). I determined that RV nucleotide substitution rates range from 1.19×10-3 substitutions/site/year (in the E1 region) to 7.52×10-4 substitutions/site/year (in the P150 region). I found that these differences between nucleotide substitution rate estimates in various RV gene regions are largely attributable to temporal sampling biases, such that datasets containing a higher proportion of recently sampled sequences will tend to have inflated estimates of mean substitution rates. Although there exists little evidence of positive selection or natural genetic recombination in RV, I revealed that RV genomes possess extensive biologically functional nucleic acid secondary structures and that purifying selection acting to maintain these structures contributes substantially to variations in estimated nucleotide substitution rates across RV genomes. Although both temporal sampling biases and purifying selection favouring the conservation of RV nucleic acid secondary structures have an appreciable impact on substitution rate estimates, I find that these biases do not preclude the use of RV sequence data to date ancestral sequences and evaluate the associated RV phylodynamics. The combination of uniformly high substitution rates across the RV genome and strong temporal signal within the available sequence data enabled me to analyse the epidemiological and demographical dynamics of this virus during these attempts to eradicate it. By implementing a generalized linear model (GLM) and symmetrical model of discretized phylogeographic spread, I was able to identify several predictive variables of geographical RV spread and detect transmission linkages between distinct geographical regions. These results suggest that, in addition to strengthened vaccination strategies, there also needs to be an increased effort to educate people about the effects of vaccination and risks of RV infection.

No description available.

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

G-quadruplex secondary structures are four-stranded globular nucleic acid structures that form in specific DNA and RNA G-rich sequences with biological significance, such as those found in human telomeres, oncogene promoter regions, replication initiation sites, and 5’- and 3’-untranslated (UTR) regions, which have been identified as novel drug targets. The non-canonical G-quadruplex secondary structures readily form under physiologically relevant ionic conditions, and exhibit great diversity in their topologies and loop conformations depending on the DNA or RNA sequences at hand. The structural diversity of these unique secondary structures is essential to their specific recognition by different regulatory proteins or small molecule compounds. A significant amount of research has been done in this field that provides compelling evidence for the existence, biological significance, and potential druggability of G-quadruplexes. In this dissertation, I explore G-quadruplex formation in the promoters of BCL2, PDGFR-β and c-Myc oncogenes and their interactions with small molecule compounds or proteins. Firstly, I investigated a newly-identified G-quadruplex (P1G4) forming immediately upstream of the human BCL2 gene, which has been found to be overexpressed in several human tumors. In this research, I have found that P1G4 acts as a transcription repressor, and that its inhibitory effect can be enriched by the G-quadruplex-interactive compound, TMPyP4. Both P1G4 and the previously reported Pu39 G-quadruplexes form independently in adjacent regions within the BCL2 P1 promoter, but P1G4 appears to play a more dominant role in repressing transcriptional activity. NMR and CD studies have shown that the P1G4 G-quadruplex appears to comprise a novel dynamic equilibrium of two parallel structures, one regular, with two 1-nt loops and a 12-nt middle loop, and another broken-stranded, with three 1-nt loops and an 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long central loop. This dynamic equilibrium of two closely-related G-quadruplex structures with a unique hairpin loop conformation may provide a specific target for small molecules to modulate BCL2 gene transcription. I also explored the 3’ end G-quadruplex that forms within the core promoter of PDGFR-β, which has also been observed to be present at abnormal levels in a variety of clinical pathologies, including malignancies. The 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively stabilized by an ellipticine analog, GSA1129, which can shift the dynamic equilibrium in the full-length sequence to favor the 3′-end G-quadruplex, and can repress PDGFR-β activity in cancer cell lines. NMR studies in combination with biophysical experiments have shown that in the wild-type extended 3ʼ-end NHE sequences, two novel intramolecular G-quadruplexes can be formed in a potassium solution, one with a 3’-flanking distant guanine inserted into the 3’-external tetrad (3’-insertion G-quadruplex), and another with a 5’-flanking distant guanine inserted into the 5’-external tetrad (5’-insertion G-quadruplex). Further investigation of the elongated PDGFR-β 3′-end sequence containing both the 5’- and 3’- flanking guanine sequences showed the formation of a combination of the two G-quadruplexes existing in equilibrium. Importantly, it was observed that GSA1129 can bind to and increase the stability of each of the end-insertion G-quadruplexes, raising their Tₘ by 25 degrees. This study highlights the dynamic nature of the 3′-end NHE sequence and the importance of identifying the proper sequence for the formation of biologically relevant G-quadruplex structures. Significantly, the dynamic nature of the 3′-end G-quadruplex suggests that it may be an attractive target for drug regulation. I then analyzed two proteins, Nucleolin and NM23-H2, which interact with the c-Myc G-quadruplex structure that forms in the proximal promoter region of the c-Myc gene; this is one of the most commonly deregulated genes in the human neoplasm. Nucleolin is known to be a transcriptional repressor for c-Myc, binding to and stabilizing the c-Myc G-quadruplex, whereas NM23-H2 is known to be a transcriptional activator that unwinds and destabilizes the c-Myc G-quadruplex. An investigation of the molecular mechanisms of the interaction between the c-Myc G-quadruplex and nucleolin showed that the minimal binding domains required for a tight binding of the protein to the c-Myc G-quadruplex are the four RNA binding domains (RBDs) of nucleolin, referred to as Nuc1234, and that the RGG domain is unnecessary for c-Myc G-quadruplex binding. The stable G-quadruplex formed within Pu27 using G-tract runs I, II, IV and V was determined to be the best substrate (Myc1245T) for nucleolin binding, showing the highest affinity. 3D NMR experiments performed on the free protein Nuc1234 and its complex with the Myc1245T G-quadruplex have shown that upon complex formation, only the disordered linker regions of the protein display significant chemical shift changes, whereas most other residues show chemical shift values similar to those of the free protein. The c-Myc G-quadruplex has three loops that flip outward in a solvent containing K⁺, according to its structure. The hypothesis for this association is that nucleolin wraps around the G-quadruplex and interacts specifically with the flipped-outward loop regions of the c-Myc G-quadruplex via its own inter-RBD linker regions, with little structural change in the RBDs themselves. A definitive determination of the 3D molecular structure of nucleolin and its complex with Myc1245T is currently in development. Biophysical and structural studies were then conducted to investigate the interactions of the protein NM23-H2/NDP kinase B with the c-Myc G-quadruplex. NM23-H2 binds to single-stranded guanine- and cytosine-rich sequences, but not to double-stranded DNA in the NHE III₁ region; the binding therefore appears structure-specific, rather than sequence-specific. Moreover, increasing concentrations of the strong G-quadruplex-interactive compound TMPyP4, a porphyrin-based drug, inhibits the binding of NM23-H2 to the NHE III₁ region; this suggests that the stabilization of the G-quadruplex hinders the recognition and remodeling function of the NM23-H2. By conducting Forster Resonance Energy Transfer (FRET) assays in combination with Circular Dichroism (CD) studies, I demonstrated that NM23-H2 can actively resolve the c-Myc G-quadruplex. Taken together, these results show that the use of small molecules to prevent NM23-H2 from binding to and resolving the NHE III₁ region G-quadruplex may have the potential to inhibit c-Myc transcription for cancer therapeutic purposes. This underlines the importance of understanding the mechanism of function operating between NM23-H2 and the c-Myc G-quadruplex. Understanding molecular mechanism between NM23-H2 and c-Myc is under investigation.

G-quadruplexes are non-canonical nucleic acid secondary structures of increasing biological and medicinal interest due to their proposed physiological functions in transcription, replication, translation and telomere biology. Aberrant G4 formation and stabilisation have been linked to genome instability, cancer and other diseases. However, the specific genes and pathways involved are largely unknown, and the work within this thesis aims to investigate this. Stabilisation of G4s by small molecules can perturb G4-mediated processes and initial studies suggest that this approach has chemotherapeutic potential. I therefore also aimed to identify cell genotypes sensitive to G4-ligand treatment that may offer further therapeutic opportunities. To address these aims, I present the first unbiased genome-wide genetic screen in cells where genes were silenced via short-hairpin RNAs (shRNAs) whilst being treated with either PDS or PhenDC3, two independent G4-stabilising small molecules. I explored gene deficiencies that enhance cell death (sensitisation) or provide a growth advantage (resistance) in the presence of these G4-ligands. Additionally, I present a validation screen, comprising hits uncovered via genome-wide screening, and also the use of this in another cell line of different origin. Sensitivities were enriched in DNA replication, cell cycle, DNA damage repair, splicing and ubiquitin-mediated proteolysis proteins and pathways. Ultimately, I uncovered four synthetic lethalities BRCA1, TOP1, DDX42, GAR1, independent of cell line and ligand. These were validated with three G4-stabilising ligands (PDS, PhenDC3 and CX-5461) using an independent siRNA approach. The latter siRNA methodology was used to screen 12 PDS derivatives with improved medicinal chemistry properties and ultimately identified SA-100-128, as a lead compound. The mechanism behind synthetic lethality with G4-stabilising ligands was explored further for DDX42, which I show has in vitro affinity for both RNA- and DNA-G4s and may represent a previously unknown G4-helicase. Also within this thesis, gene deficiencies that provided a growth advantage to PDS and/or PhenDC3 as uncovered by genome-wide and focused screening were explored. These showed enrichment in transcription, chromatin and lysosome-associated genes. The resistance phenotype of three gene deficiencies, TAF1, DDX39A and ZNF217 was further supported by additional siRNA experiments. Overall, I satisfied the primary aims and established many novel synthetic lethal and resistance interactions that may represent new therapeutic possibilities. Additionally, the results expand our knowledge of G4-biology by identifying genes, functions and subcellular locations previously not known to involve or regulate G4s.