This thesis is concerned with the synthesis of diazo compounds for the introduction of various functional groups on the surface of polymers by means of carbene insertion and diazonium coupling. Characterisation and investigation of their properties were conducted. A library of functionalised polystyrene beads containing pyridine rings has been established for the coordination of metal ions and metal complexes. The pyridyl system on the surface has demonstrated its capability to bind with zinc complexes of bis(thiosemicarbazones) and release the corresponding copper complexes upon transmetallation. A spiropyran derivative has been introduced onto the surface of polystyrene and polyethylene terephthalate. The chromophore on both polymers exhibited photoswitchability as determined from colour change and wettability. Polystyrenes with either pyridine or spiropyran units have been investigated for reversible immobilisation of bioactive species. Materials coated with penicillin V gave significant inhibition zones in antibacterial assays, showing efficacy in drug delivery. Finally, a range of diazo compounds with different substituents has been synthesised and their thermal stabilities have been assessed by differential scanning calorimetry technique.

Eukaryote DNA is organised into the more compact nucleosome by wrapping 147bp of DNA around a histone octamer core. The N-terminal tails of the histones protrude through the DNA and can be modified by a variety of enzymes. Acetylation of Histone 4 Lysine 16 (H4K16ac) is an important modification associated with an increase in transcription, and in flies is an important component of the doseage compensation system. It is also unique amongst histone modifications in that it has been directly associated with chromatin decompaction. H4K16ac has been linked to development through its Histone Acetyltransferase, MOF. Deletion of MOF in mice leads to mass chromatin defects, and embryonic lethality prior to the blastocyst stage. I set out to understand the role of H4K16ac in differentiating Embryonic Stem cells (ES cells) and chromatin compaction in vivo. I generated a ChIP-seq profile for H4K16ac in undifferentiated ES cells, and after 3 days of retinoic acid (RA) differentiation. This revealed an association of H4K16ac with the promoters of transcribed genes in pluripotent ES cells, followed by loss H4K16ac on ES cell specific genes and gain of the modification on differentiation specific genes. There were some silent genes in ES cells, however, which were acetylated on their promoters. Through this study I also found that H4K16ac and MOF mark active enhancers in ES cells, along with H3K4me1 and H3K27Ac and p300. H4K16ac did not mark a known regulatory region in limb cells, and it is possible that it marks active enhancers only of ES cells. Furthermore, I looked at the compaction state large regions (>100kb) which lost H4K16ac upon differentiation by FISH, to determine if loss of H4K16ac could predict compaction. The regions selected showed no change in compaction state between UD and D3 cells, meaning that loss of H4K16ac does not directly lead to chromatin compaction in vivo. However loss of H4K16ac may be necessary for any subsequent compaction, or the change in compaction may take place at nucleosomal level. Finally, I attempted both to overexpress and reduce the level of MOF in ES cells. I was unable to manipulate the level of MOF in this cell type in either direction; expression of endogenous MOF was silenced after very little time, and stable MOF shRNA cell lines showed no reduction in levels of MOF. Therefore, potentially, dosage of MOF/H4K16ac in this cell type is critical. This study may help to understand the significance of H4K16ac in ES cell differentiation and chromatin compaction.

This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge on the ocr molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues were progressively introduced into pocr was generated. A high-throughput in vivo selection assay was developed and validated by assessing the antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones that displayed antirestriction activity. These mutants were purified and in vitro characterisation confirmed these mutants as displaying the minimum number of acidic residues deemed critical for the activity of ocr. This in vitro process effectively simulated the evolution of the charge mimicry of ocr. Moreover, we were able to tune the high-throughput assay to different selection criteria in order to elucidate various levels of functionality and unexpected changes in phenotype. This approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are required for protein expression, solubility and function proceeding to a fully functional antirestriction protein.

No description available.

The purpose of the present study was to determine whether computer-based cognitive bias modification (CBM) procedures could alter appraisal style toward viewing events from a big picture perspective and thereby influence emotional reactivity. Big picture appraisal entails viewing difficult situations and one's reactions to them in terms of a larger context. Appraisal training was implicit in that participants completed a series of vignettes, framed as a reading comprehension task, which trained either a big picture perspective or a personal/evaluative focus. When subsequently confronted with novel vignettes, participants produced interpretations that were consistent with assigned training condition. In addition, participants trained in big picture as compared to personal/evaluative appraisal subsequently demonstrated less emotional reactivity to a stressful task. / text

Once historically widespread throughout the Willamette and Umpqua River drainages, the Willamette Oregon chub is now restricted to scattered populations along 15 miles of the Middle Fork of the Willamette River whereas the Umpqua Oregon chub is still widely distributed. The decline in the Willamette drainage was more severe because changes in the physical and biological habitat were more severe when compared to the Umpqua drainage. The Willamette Oregon chub may be more sensitive to environmental degradations than the Umpqua Oregon chub. The difference in sensitivity to degradation is a result of ecological differences between Willamette and Umpqua Oregon chub. Willamette Oregon chub inhabited areas with primarily very little if any current, fed in the water column, and spawned in aquatic vegetation. Umpqua Oregon chub inhabited areas with primarily slow water velocities, fed on benthic prey, and spawned over gravel substrate. Because Willamette Oregon chub inhabit slackwater habitats they may be more sensitive to water velocity increases and exotic species, whereas Umpqua Oregon chub have a habitat refuge in relatively faster water velocity habitats. Willamette Oregon chub larval stages are described and illustrated. The following combination of characters distinguish Willamette Oregon chub larvae from other cyprinid larvae found in the Willamette drainage: 33-37 myomeres, preanal length of 52-61%, and absence of snout pigment. Willamette Oregon chub larvae generally congregated in nearshore areas, in the upper layers of the water column, in shallow water. They fed primarily in the water column, and ate primarily rotifers and cladocerans. / Graduation date: 1989

No description available.

The effectiveness of imagery utilizing counterconditioning in the modification of attitudes toward elderly persons was explored. Ss were 162 introductory psychology students. Ss were divided into five groups with one group serving as a no-treatment control. The remaining four groups were instructed to imagine one of four types of scenes: I) reinforcing imagery scene with subjects instructed to picture themselves as the old man, 2) reinforcing imagery scene only, 3) neutral imagery scene with subjects instructed to picture themselves as the old man, 4) neutral imagery scene only.The criterion measure was post-test scores on an attitude questionnaire. An analysis of covariance treatment effects when the effects of the pre-test were held constant. No significant correlations were found between number of times Ss reported practicing the scenes and difference scores from pre-test to post-test. No support was given to the counterconditioning hypothesis of attitude change.

No description available.

Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N-methylguanosine to 4-demethylwyosine by the radical S-adenosyl-l-methionine enzyme TYW1. When these studies were initiated, the substrate of TYW1 was unknown. Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, ¹³C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct. Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5'-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group. The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate. The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro. It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of N-methylguanosine to 4-demethylwyosine.