For many human protein-coding genes, alternative cleavage and polyadenylation (APA) of pre-mRNA generates distinct 3' untranslated regions (3'UTRs) with differing regulatory potential. Widespread 3'UTR shortening via APA occurs in proliferative cell states, including cancer, where it can lead to oncogene overexpression. There has therefore been significant interest in identifying factors which influence poly(A) site choice in different physiological states. The multi-subunit human cleavage factor I complex (CFIm), a core component of the mammalian pre-mRNA cleavage machinery, has been identified as a potential master regulator of APA, as its depletion leads to widespread 3'UTR shortening. However, mechanistic understanding of how CFIm influences poly(A) site selection, and how its activity is regulated, is lacking. In this work, gene editing was used to generate cell lines with substantial, permanent depletion of the 25 kDa or 68 kDa subunits of CFIm (CFIm25 and CFIm68), which exhibited the expected 3'UTR shortening for representative transcripts. Reversal of this 3'UTR shortening by CFIm25 or CFIm68 re-expression provided the basis for a complementation assay, which allowed various aspects of CFIm25 and CFIm68 function to be investigated in vivo. The capacity of CFIm25 to recognise UGUA RNA sequences was shown to make an important contribution to poly(A) site selection transcriptome-wide, and a novel function for the C-terminal arginine/serine-rich (RS domain) of CFIm68 in poly(A) site selection was identified. The potential contribution of CFIm post-translational modification (PTM) to APA regulation was also explored. Novel acetylation sites on CFIm25 and CFIm68 were identified, as well as extensive serine phosphorylation in the CFIm68 RS domain. Complementation analysis revealed that phosphomimetic mutations in this RS domain inhibited distal poly(A) site selection, suggesting a potential role for CFIm68 phosphorylation in APA regulation. Taken together, the findings presented here provide insights into several important determinants of CFIm function, and the complementation assay developed provides a useful tool for future investigations.

>Magister Scientiae - MSc / We report the successful syntheses of a family of tetradentate N-donor pyridyl and quinolyl-imine ligands N1,N2-bis((pyridin-2-yl)methylene)ethane-1,2-diamine (L1), N1,N3- bis(pyridin-2-ylmethylene)propane-1,3-diamine (L2), N1,N4-bis(pyridin-2-ylmethylene) butane-1,4-diamine (L3), N1,N2-bis((quinolin-2-yl)methylene)ethane-1,2-diamine (L4), N1,N3-bis(quinolin-2-ylmethylene)propane-1,3-diamine (L5) and N1,N5-bis(pyridin-2- ylmethylene)pentane-1,5-diamine (L6). All the ligands were fully characterized by FT-IR, 1H and 13C NMR, GC-MS, Elemental analysis, UV-Vis and TGA. We report for the first time the thermogravimetric analysis of N1,N2-bis((pyridin-2-yl)methylene)ethane-1,2-diamine (L1) and N1,N2-bis((quinolin-2-yl)methylene)ethane-1,2-diamine (L4). The tetradentate N-donor pyridyl and quinolyl-imine ligands were subsequently utilised to synthesise neutral mononuclear and cationic homobimetallic ruthenium(II) complexes and new bimetallic palladium(II) complexes using the appropriate metal precursors. The ruthenium(II) complexes were evaluated for the oxidative cleavage of styrene using a Sharpless biphasic solvent system (CCl4:CH3CN:H2O) and sodium periodate (NaIO4) as the cooxidant. The bimetallic palladium(II) complexes were evaluated for their catalytic activity towards the standard Heck coupling reaction. The ruthenium(II) catalysts exhibited efficient catalytic activity, yielding conversions of 69-77%. The palladium(II) catalysts showed an overall low catalytic activity of 41-49 % conversion and analysed by GC.

Welding is an essential process in many industries for both the production and repair of engineering plant, notably pressure vessels and piping. However, welding processes cause large magnitudes of residual stress to be induced within the structure. Residual stress can be defined as a stress that exists in a material when it is under no primary loading. Whilst residual stresses can be reduced by post weld heat treatment, such treatments are not always possible, and so high residual stresses can remain in serviceThe current methodology for evaluating fracture toughness from specimens, particularly if these contain weld residual stresses is presented in BS7448-1997. This method relies on the assumption that the effect of residual stress on fracture toughness measurements can be negated by the application of a local compression, to the ligament ahead of the pre-crack in the test specimen. Recent research has investigated the validity of this assumption. The results suggest that, far from being removed, the residual stresses are modified or even enhanced via local compression. This can reduce the value of measured fracture toughness below its true value. In order to ensure the validity of fracture toughness measurements in materials that contain residual stress, a more robust method for its quantification is developed.The aim of this project was to extend current understanding regarding the magnitude and distribution of residual stresses retained in standard fracture mechanics specimens removed from welds and the consequent effects of these stresses on measured fracture toughness, both in terms of the crack driving force and crack-tip constraint. Furthermore, the project aimed to derive improved methods for the quantification of valid values of fracture toughness from laboratory specimens containing residual stresses. This was achieved via a combination of analytical and experimental work.The effect of specimen extraction on the level of retained residual stress in specimens extracted from non stress-relieved welds was investigated using parametric finite element analyses. Simplified methods to quantify the levels of residual stresses in fracture mechanics specimens removed from welds and their significance, in terms of contribution to crack driving force, are proposed.The influence of residual stresses on the measured fracture toughness properties of ferritic pressure vessel steel, tested in the cleavage fracture regime, has also been studied. A refined method of out-of-plane compression was devised and used to generate significant residual stresses in three-point bend specimens. This method was then used experimentally, alongside supporting elastic-plastic analyses, to quantify the effects of the residual stresses on fracture toughness in terms of both crack driving force and crack-tip constraint in geometrically high and low constraint specimens. A method whereby fracture toughness data, obtained from specimens containing residual stresses, can be corrected to provide valid fracture toughness properties using constraint based fracture mechanics alongside a simple fracture model has been proposed. The main conclusions from the work are as follows. Significant weld residual stresses have been shown to be retained in certain laboratory specimens post extraction from non stress-relieved welds. The magnitude and distribution of retained residual stress has been shown to be dependant on: • Material yield and flow properties • Specimen size; where larger specimens are more likely to retain significant levels of residual stress than smaller specimens • Specimen type; either compact tension (CT) or single edge notched bend (SENB), where there is a tendency for specimens to retain higher relative levels of residual stress in the directions of their largest dimensions; i.e. bend specimens retain more residual stress along their length than CT specimens and CT specimens retain more residual stress across their width than bend specimens • Extraction location, e.g. full thickness, near surface, mid-thickness etc. The stress partitioning method has been shown to provide a useful estimating approach for assessing the levels of residual stress retained in fracture mechanics specimens extracted from non stress-relieved welds in certain orientations.Retained residual stresses have been shown to affect both crack driving force and crack-tip constraint in both low and high geometrically constrained 50mm bend specimens manufactured from A533B ferritic steel. The residual stress has been shown to dominate the level of crack-tip constraint condition over and above the geometric and loading factors during the early stages of loading. The effects of residual stress on crack driving force and crack-tip constraint have been shown to result in fracture loads and, therefore, measured fracture toughness values that vary widely from those to be expected in the material under small-scale yielding conditions; i.e. if a standard specimen were to be tested containing no residual stress. Two-parameter (J-Q) fracture mechanics has been shown to provide a valid approach for quantifying fracture toughness properties from high and low constraint specimens, with and without residual stresses, with all data being shown to be consistent with a J-Q failure locus for a given level of cleavage probability.

The work presented in this dissertation has focused on synthesizing complexes of relevance to dioxygenase enzymes that oxidatively cleave aliphatic carbon-carbon bonds. The goal of this research was to elucidate mechanistic aspects of the activation of aliphatic carbon-carbon bonds towards cleavage by reaction with oxygen, and also investigate the regioselectivity of these reactions. The oxidative cleavage of a variety of enolizable substrates has been explored by utilizing several transition metal complexes supported by an aryl-appended tris(pyridylmethyl)amine ligand. In order to probe the widely-accepted “chelate hypothesis” for how changes in regiospecificity are achieved as a function of metal ion, we synthesized the compound [(6Ph2TPA)Fe(PhC(O)COHC(O)Ph)]OTf. Based on UV-vis and IR spectroscopy, the acireductone enolate was found to bind via a six-membered chelate ring. By comparison with the reactivity of [(6Ph2TPA)Ni(PhC(O)COHC(O)Ph)]ClO4, we determined that the chelate hypothesis was an insufficient explanation of the observed regioselectivity. Rather, ferrous ion-mediated hydration of a vicinal triketone intermediate was the key factor in determining the regioselectivity of the C-C cleavage reaction. We have developed a high-yielding synthetic route to protected precursors of C(1)H acireductones. Preparation of the complexes [(6Ph2TPA)M(PhC(O)COCHOC(O)CH3)]ClO4 (M = Fe, Ni) followed by judicious choice of deprotecting conditions allowed us to investigate the oxygen reactivity of a mono-nuclear complex with a dianionic acireductone substrate for the first time. This provides a promising strategy to continue investigations of complexes of relevance to the enzyme- substrate adduct of the acireductone dioxygenases. Divalent late first-row transition metal complexes have been used to investigate some new strategies for the activation of dioxygen and subsequent cleavage of C-C bonds. We have utilized photoreduction of a Ni(II) center to generate a highly O2-reactive Ni(I) fragment that leads to cleavage of a chloro-diketonate substrate. Additionally, we have found a Cu(II)-mediated thermal cleavage of chloro-diketonate substrates at room temperature. This reaction is interestingly accelerated by the addition of a catalytic amount of chloride ion.

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Thesis (Ph. D.)--Miami University, Dept. of Botany, 2008. / Title from second page of PDF document. Includes bibliographical references.

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Expression of the cellular prion protein (PrPC) from the PRNP gene is crucial for the development of a group of fatal neurodegenerative disorders called prion diseases. During prion infection a misfolded protein homologue of PrPC, PrPSc causes further misfolding on interaction with native PrPC molecules. PrPSc is highly resistant to proteinase K and aggregation of this protein is considered a hallmark of infection. Sheep are considered a model of natural infection and susceptibility to scrapie in sheep is defined by polymorphisms in the PRNP gene. It is still not fully understood how these polymorphisms regulate the conversion process or which other co-factors are involved. One such factor may be the truncation of PrPC via proteolytic processing in the form of two main cleavage events, known as α- and β-cleavage. In sheep α-cleavage cuts at amino acid 115, creating two truncated proteins C1 and N1 and represents the main cleavage event in healthy brain. β-Cleavage creates a longer C-terminal fragment, C2 and corresponding N-terminal fragment N2, cutting around amino acid 92 in sheep. Truncated forms of PrPC have been shown to represent around 50 % of total residual PrP in brain and may be an important determinant of disease through both decreasing the amount of full length PrPC available for conversion and through functions associated with the truncated fragments. The research presented has shown that increased production of an α-cleavage fragment C1 in brain is associated with TSE resistant genotype ARR/ARR, while the presence of C2 fragment is affiliated with scrapie susceptible PRNP genotypes in brain. There was no difference in the levels of full length PrPC in these genotypes suggesting that PrP expression does not directly correlate to susceptibility in this model. To assess if PrPC fragments could affect the conversion during disease in-vitro fibrillisation assays were performed using novel truncated recombinant proteins. These truncated proteins, although not thought to convert to PK resistant PrPSc during disease, can form amyloid fibrils. However, these fibrils appear to be less neurotoxic when compared to fibrils produced by full length PrPC. Only the truncated fragments derived from the ARR allele inhibit in-vitro fibrillisation of other allelic PrPC variants. Furthermore, treatment of infected cells in culture with recombinant C1ARR led to a decrease in the formation of disease associated PrPSc. In conclusion, genetic variations in levels of PrP truncated fragments may add to the complexity of genetic determinants of prion disease. In parallel with polymorphism-dependant conversion abilities, varying α-cleavage of ovine PrPC may help to explain genetic resistance in sheep. The inhibitory effects of C1, illustrated in-vitro may represent a therapeutic avenue in the treatment of prion disease.