No description available.

<p>DNA interstrand cross-links (ICLs) constitute one of the most severe types of DNA damage. ICLs covalently tether both strands of duplex DNA, preventing unwinding and polymerase access during replication and transcription. This obstruction is exploited in cancer chemotherapy since it leads to replication fork collapse, double strand breaks (DSBs), and cell death. Mechanistic understanding of how eukaryotic cells repair these specific lesions, however, is still in its infancy. It is understood that ICL repair consists of a multitude of intersecting and connecting repair pathways that rely on interplay between critical protein factors. Interestingly, Pso2 has been identified as an integral component of the ICL repair pathway in <em>Saccharomyces cerevisiae</em>. Pso2 is a yeast nuclease from the β-CASP family of proteins that function predominantly in the repair of ICLs. It has been recognized as the only protein that does not serve a redundant function in any other DNA repair pathway. It remains unclear how the ICL repair pathway generates DNA intermediates suitable for high fidelity repair dependent on Pso2 nuclease activity. Here we show that Pso2 possesses structure-specific endonuclease activity that may be essential to its role in ICL repair. Direct <em>in vitro</em> activity assessment of the protein on a site-specific ICL proved to be inconclusive due to the heat-labile nature of the cross-linking agent employed. <em>In vitro </em>activity testing was also performed on various substrates resembling intermediates generated during ICL repair. Biochemical analysis demonstrated that Pso2 cleaves hairpins, stem loops, heterologous loops, and symmetrical bubbles. Although the precise cleavage sites vary between substrates, Pso2 demonstrates preference for the single- to double-stranded junction in the DNA backbone, with similar activity to that previously demonstrated for its human homologue, Artemis. This specific endonuclease activity is stimulated by increased concentrations of phosphate. Through two-dimensional gel electrophoresis, the presence of unique DNA intermediates generated in response to ICL damage <em>in </em><em>vivo </em>was also monitored. Results suggest the generation of hairpin-like intermediates that resemble those tested <em>in vitro</em>. These intermediates persist in the absence of Pso2 but are resolved by exogenous addition of control endonucleases. Our findings expand on previous data that established hairpin-opening activity for this protein and suggest that the structure-specific endonuclease activity demonstrated by Pso2 is important for ICL repair. We anticipate that Pso2 acts on a hairpin-containing DNA substrate in the ICL repair pathway and the resolution of this intermediate is uniquely dependent on Pso2 for the effective repair of ICL damage in yeast. Taking into consideration the current models of ICL repair, both in yeast and humans, possible roles for Pso2 have been described. Achieving a complete mechanistic perspective of this pathway is critical for the therapeutic exploitation of the human homologue, SNM1A. Implications include the potential inhibitory target for increased efficacy of chemotherapy with cross-linking agents.</p> / Master of Science (MSc)

G-quadruplexes (GQs) are highly stable noncanonical nucleic acid structures that form in the DNA of human cells and play fundamental roles in maintaining genomic stability and regulating gene expression. These unique structures exert broad influence over biologically important processes and can modulate cell survival and human health. In fact, mutations, hyper-stability, and dissociation of GQs are implicated in neurodegenerative disease, mental retardation, premature-aging conditions, and various cancers. As such, GQs are novel drug targets. GQ-targeting therapeutics are developed to influence the folding and genetic interactions of GQs that are implicated in diseased states. To do so requires a greater understanding of GQ structure and dynamics and molecular dynamics (MD) simulations are well suited to provide these fundamental insights. Previous MD simulations of GQs have provided limited information due to inaccuracies in their models, namely the nonpolarizable nature of their force fields (FFs). The cutting-edge Drude polarizable FF models electronic degrees of freedom, allowing charge distribution to change in response to its environment. This is an important component for modeling ion-ion and ion-DNA interactions and can influence the overall stability of GQ structures. The work herein employs the Drude polarizable FF to 1) describe the role of electronic structure on the dynamics and folded stability of GQs, 2) determine the impact of ion interaction on GQ stability, and 3) characterize the role of G-hairpin motifs in GQ intermediates. Such fundamental investigations will help clarify GQs role in healthy and diseased states and transform our understanding of noncanonical DNA, improving human health, therapeutic design, and fundamental science. / Doctor of Philosophy / Human health and disease are influenced by unique nucleic acid structures called G-quadruplexes (GQs). GQs form when DNA or RNA fold into a square-shaped structure that is stabilized by ion interactions and special hydrogen bonding patterns. These GQ structures exert broad influence over normal biological processes, but also play a role in neurodegeneration, intellectual disabilities, premature-aging conditions, and various cancers, many of which are chemotherapeutic resistant. As such, modulating GQ structures, or their interactions with proteins, is a promising therapeutic approach. However, a greater understanding of GQ folding, folded structure, and interactions with other biomolecules is needed to do so. Computational techniques such as molecular dynamics (MD) simulations use experimental data and fundamental biophysics to gain new insights on these properties and inform novel drug design. In this project, we explored the dynamics of several distinct GQ structures and folding intermediates with state-of-the-art MD simulation methods. In doing so, we provided new insight on their structural features as well as their interactions with extended DNA sequences and different ion types, which serve as fundamental information for future structural or computer-aided drug design studies.

Epigenetics are stable heritable traits that are not a result of the DNA sequence. Epigenetic modification of DNA cytosine plays a role in development and disease. The covalent bonding of a methyl group or a hydroxymethyl group to the 5-carbon of cytosine epigenetically modifies cytosine to 5-methylcytosine or 5-hydroxymethylcytosine. Upon PCR amplification, the bisulfite treatment of DNA converts unmethylated cytosine to thymine, while 5-methylcytosine, 5-hydroxymethylcytosine, and other bases remain unchanged. The resulting sequences can be mapped to a reference genome; however, this can be challenging due to sequencing technology complexity, low sequence complexity, and biases and errors introduced with bisulfite treatment. Once the short read is mapped, the identity of 5-methylcytosine or 5-hydroxymethylcytosine can be determined by comparing the mapped read to the aligned reference genome. Bisulfite DNA read mapping is characterized by mapping performance as low as 40%. This research improves bisulfite short read mapping quality. First, reads generated from the bisulfite hairpin PCR protocol are used to study mapping failure and solutions. A read may not map to the genome; it may map uniquely, or it may map to multiple locations. Sequence complexity correlates with these mapping categories. The hairpin protocol allows for a recovery, in some cases, of the original untreated read, and mapping this read with the regular read mapper Bowtie2 improved mapper performance by 10%. New bisulfite read mapping software called BisPin was created that calls BFAST (BLAT-like Fast Accurate Search Tool) for mapping. BisPin resolves ambiguously mapped reads with a rescoring strategy, which yields a statistically significant improvement. BFAST-Gap for Ion Torrent reads was developed, since Ion Torrent machines are less expensive than Illumina machines and since Ion Torrent reads are longer. There are few mappers for Ion Torrent data. BFAST-Gap uses homopolymer run length for contextual gap penalty functions, since homopolymer runs cause errors in Ion Torrent reads. In conjunction with BisPin, this software performed well on real and simulated bisulfite Ion Torrent data and Illumina data. InfoTrim, a read trimmer with an entropy term, was developed with competitive results. / Ph. D.

ΔPhe an analogue of a coded amino acid phenylalanine (Phe) residue but with double bond between Cα and Cβ atoms, is one of the well studied residue among all the dehydro amino acids, as a conformation constraining amino acid. Due to the presence of double bond Cα=Cβ, and consequent conjugation of ΔPhe ring electrons with Cα=Cβ double bond, ΔPhe gains conformation restricting (constraining) characteristics compared to coded amino acid Phe. ΔPhe which assumes an achiral residue has all its atoms restricted to an approximate plane. Apart from the conformation constraining property, the designer friendly ΔPhe residue has its ability to i) engage in side chain aromatic interactions ii) act as nuclei for C-HLO/N-HLπ weak interactions involving the side chain and/ or backbone atoms, and iii) acquire ambidextrous conformation as observed in many model peptides. It is these properties, which makes ΔPhe, a residue of intense research in the field of de novo protein secondary and super secondary design. Analysis of solid state and solution state structures of containing ΔPhe residues suggests that ΔPhe, in general induces β-bend in short peptides and 310-helical conformation in longer peptides (>4).

This thesis is an experimental study of turbulent flows over smooth and rough walls in a channel that consists of an upstream parallel section to produce a fully developed channel flow and a diverging section to produce an adverse pressure gradient (APG) flow. The roughness elements used were two-dimensional square ribs of nominal height k = 3 mm. The ribs were secured to the lower wall of the channel and spaced to produce the following three pitches: 2k, 4k and 8k, corresponding to d-type, intermediate and k-type rough walls, respectively. For each rough wall type, the ribs were inclined at 90°, 45° and 30° to the approach flow. The velocity measurements were performed using a particle image velocimetry technique. The results showed that rib roughness enhanced the drag characteristics, and the degree of enhancement increased with increasing pitch. The level of turbulence production and Reynolds stresses were significantly increased by roughness beyond the roughness sublayer. It was observed that the population, sizes and the level of organization of hairpin vortices varied with roughness and more intense quadrant events were found over the smooth wall than the rough walls. APG reinforced wall roughness in augmenting the equivalent sand grain roughness height, turbulence production and Reynolds stresses. APG also reduced the sizes of the hairpin packets but strengthened the quadrant events in comparison to the results obtained in the parallel section. The secondary flow induced by inclined ribs significantly altered the distributions of the flow characteristics across the span of the channel. Generally, the mean flow was less uniform close to the trailing edge of the ribs compared to the flows at the mid-span and close to the leading edge of the ribs. The Reynolds stresses and hairpin packets were distinctly larger close to the trailing edge of the ribs. Rib inclination also decreased the drag characteristics and significantly modified the distributions of the Reynolds stresses and quadrant events. In the parallel section, the physical sizes of the hairpin packets were larger over 45° ribs whereas in the diverging section, the sizes were larger over perpendicular ribs.

This thesis is an experimental study of turbulent flows over smooth and rough walls in a channel that consists of an upstream parallel section to produce a fully developed channel flow and a diverging section to produce an adverse pressure gradient (APG) flow. The roughness elements used were two-dimensional square ribs of nominal height k = 3 mm. The ribs were secured to the lower wall of the channel and spaced to produce the following three pitches: 2k, 4k and 8k, corresponding to d-type, intermediate and k-type rough walls, respectively. For each rough wall type, the ribs were inclined at 90°, 45° and 30° to the approach flow. The velocity measurements were performed using a particle image velocimetry technique. The results showed that rib roughness enhanced the drag characteristics, and the degree of enhancement increased with increasing pitch. The level of turbulence production and Reynolds stresses were significantly increased by roughness beyond the roughness sublayer. It was observed that the population, sizes and the level of organization of hairpin vortices varied with roughness and more intense quadrant events were found over the smooth wall than the rough walls. APG reinforced wall roughness in augmenting the equivalent sand grain roughness height, turbulence production and Reynolds stresses. APG also reduced the sizes of the hairpin packets but strengthened the quadrant events in comparison to the results obtained in the parallel section. The secondary flow induced by inclined ribs significantly altered the distributions of the flow characteristics across the span of the channel. Generally, the mean flow was less uniform close to the trailing edge of the ribs compared to the flows at the mid-span and close to the leading edge of the ribs. The Reynolds stresses and hairpin packets were distinctly larger close to the trailing edge of the ribs. Rib inclination also decreased the drag characteristics and significantly modified the distributions of the Reynolds stresses and quadrant events. In the parallel section, the physical sizes of the hairpin packets were larger over 45° ribs whereas in the diverging section, the sizes were larger over perpendicular ribs.

No description available.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5 ' and 3 ' UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5 ' end (2-21 nt) of the 5 ' UTR. Nucleolin binds the hairpin loop structure in the 5 ' UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3 ' UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)(n), <= 26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

Ziel dieser Arbeit war die Entwicklung eines Twinribozyms, das durch zweifache Spaltung und anschließende zweifache Ligation den Fragmentaustausch innerhalb einer RNA-Substratsequenz katalysiert. Als Basis für dieses Konzept diente das Hairpinribozym, das durch die Einführung einer zusätzlichen Helix derart verändert wurde, dass die Verknüpfung zweier Einheiten in einem Twinribozym möglich wurde. Weitere Sequenz- und Strukturoptimierung führte zu zwei Einzelmotiven mit hoher Substratselektivität und verbesserter Kofaktorakzeptanz. Durch die Verwendung von 5-Benzylmercapto-1H-tetrazol als Aktivator beim Standard-Phosphoramidit-Verfahren konnte die chemische RNA-Synthese in einem Umfang verbessert werden, der die Synthese eines Twinribozyms mit einer Länge von 141 Nukleotiden und beidseitiger Spaltaktivität erlaubte. Auf enzymatischem Weg wurde ein Twinribozym HP-TW5 synthetisiert, das aus einem 45-mer RNA-Substrat ein 16-mer Fragment herausschneidet und durch ein 20-mer Reparaturfragment ersetzt. Das nach Ligation in bis zu 30 % Ausbeute entstandene 49-mer Produkt wurde durch Sequenzanalyse eindeutig nachgewiesen. Auf diese Weise konnte die Reparatur einer Deletionsmutation in einer Modellreaktion erfolgreich durchgeführt werden. Die Twinribozym-vermittelte RNA-Reparatur bietet damit einen neuartigen Zugang zu gentherapeutischen Anwendungen. / Aim of this work was the development of a twin ribozyme that catalyses the fragment exchange within a RNA substrate sequence by twofold cleavage and subsequent twofold ligation. The hairpin ribozyme, serving as basis for this concept, was changed by introduction of an additional helix in a way, that connection of two units in a twin ribozyme became possible. Further sequence and structure optimization led to two different motifs with high substrate selectivity and improved activity under different cofactor conditions. By application of 5-Benzylmercapto-1H-tetrazole as activator in the standard phosphoramidite method, chemical RNA synthesis could be highly improved, allowing synthesis of a Twinribozyme with a length of 141 nucleotides and cleavage activity of both units. The enzymatically synthesised twin ribozyme HP-TW5 was used to cut out a 16-mer fragment from a 45-mer RNA substrate and replace it with a 20-mer repair fragment. The chemical identity of the 49-mer repair product, resulting from ligation in up to 30 percent yield, was proven by sequence analysis. With this model reaction, the repair of a deletion mutation could be successfully demonstrated. Twin ribozyme-mediated RNA-repair offers access to new applications in gene therapy.