DNA photocleaving reagents are a unique class of molecules that display the ability to cleave DNA, causing strand breaks, upon exposure to an irradiation source. In terms of biological applications, achieving excitation through a two-photon absorption event provides for unique benefits that can be useful in such applications as photodynamic therapy and cell viability studies. Thus, this thesis pertains to the study of a class of photocleaving reagents that have been shown to become excited through a twophoton process during irradiation with a pulsed femtosecond laser at 775 nm. N-(Alkoxy)pyridinethiones were selected as possible oxygen-based radical generators upon irradiation at two-photon wavelengths. Experiments were carried out with pBR 322 plasmid DNA to determine if these N-(alkoxy)pyridinethiones could cause strand cleavage and if so how efficient they are in doing so. Several compounds were found to be effective DNA strand cleavers when irradiated at two-photon wavelengths, displaying the utility of two-photon excitation in biological studies. Rationale is suggested for the observed variation in cleaving efficiency based on inherent properties of the generated radicals. A second study was done to measure the two-photon cross section of the compound N-(anthracenoyloxy)pyridinethione. The two-photon cross section was found by measuring the fraction of substrate remaining after specific periods of femtosecond laser irradiation at 775 nm, and the two-photon cross section was found to be 0.051 GM.

I examine the relationship between social structural factors and political behavior by applying the concept of social cleavage in American society. Lipset and Rokkan (1967) developed the concept of social cleavage to explain the influence of social structure on political behavior in the 1960s. They suggest that social cleavage emerged in Western Europe in the 1920s and persisted until the 1960s. Some scholars claim that the influence of social group membership is not as influential in predicting voting behavior in elections as it was in the 1960s, while other scholars argue that social cleavages are still important in explaining individuals’ choices in elections. Additionally, many scholars believe that issue-based factors reduce the influence of social structure on voting behavior. I first analyze the voting trend of classes, religious groups, and regions, and their magnitude of cleavage since 1980. Second, I examine the influence of economic and cultural factors on Presidential voting. Third, I estimate the relative size of the effects of economic and cultural factors on Presidential voting. Fourth, I demonstrate the influence of economic and economic factors on social cleavages. The findings show that social group membership and geographical residence are significant factors in Presidential elections between 1980 and 2008. Political cleavage based on religious group membership is the greatest. Voters also have more distinctive political preferences based on micro-regional residence compared to macro-regional residence. The binary logistic regression analysis showed that economic and cultural factors are significantly associated with Presidential elections between 1984 and 2008, and that the magnitude of social cleavage changed when economic and cultural variables were included.

Intact and fractured rock samples were studied in the laboratory in order to understand more fully the mechanism of closure of fractures subjected to high confining stresses and the resultant effect on specimen permeability. Confining stresses applied to the specimens ranged from 3.0 to 20.0 MPa, and the closure of fractures was observed by monitoring the change in the hydraulic conductivity of the specimens. Test results suggest that some resealing may occur due to crushing and realignment of mineral grains along a fracture surface. The closure of fractures is dependent upon the strength of the rock mass, the physical nature of the fracture, and the fluid pressure present in the fracture. Fracture closure is highly time dependent, and a number of nonlinear pressure flow relationships have been identified. These deviations are thought to represent two fundamentally different processes, the most important of which are turbulence in the flow and fracture expansion.

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Homogenous glycoforms of glycoproteins are one of the primary targets in glycobiology to allow for the determination of glycoprotein function and to create glycoprotein mimetics for use as therapeutic agents. Although N- and O-linked glycosylation is preferred in nature, S-linked glycoproteins are attractive synthetic targets due to their enhanced chemical stability and enzymatic resistance. This thesis reports a novel convergent method for the synthesis of S-linked glycoproteins through the site-specific ligation of 1-glycosyl thiols to olefin containing proteins. This strategy employs unnatural amino acid incorporation for the introduction of L-homoallylglycine (L-Hag) into proteins and free-radical addition hydrothiolation chemistry, under conditions mild enough to preserve protein activity. The unique reactivity profile of L-Hag, which has an olefinic side-chain, compared with the natural amino acids characteristically found in proteins, allows for a chemoselective chemical reaction. At the outset, methodology was optimised using Hag as a model system. The reaction was then successfully applied to proteins with different structures, for precise, site-selective glycoconjugation at single and even multiple sites. Whilst investigating the above-mentioned photochemical modification of proteins, an unexpected photo-induced site-selective protein cleavage reaction localised to the TIM-barrel proteins from family 1 of glycosylhydrolases was discovered. Although proteins are known to be affected by UV irradiation, examples of clean, site-selective photocleavage without the use of a cleaving agent are rare. Remarkably, the β-glycosidase from Sulfolobus solfataricus (SsβG) was found to form two daughter fragments following UV irradiation (240–308 nm). Alterations at and alaninescanning of a sphere of residues around the cleavage site were used to establish which residues were essential for the reaction to occur and extensive analysis of the fragments allowed a reaction mechanism to be proposed. The propensity of other proteins to undergo photo-induced cleavage was also investigated. In addition, the biotechnological utility of the aforementioned photocleavage reaction has been applied to the development of a cleaved-tag affinity purification method using a GFPSsβG fusion as the model protein.

Class III Semaphorins (SEMA3) comprise a family of chemokines that have been implicated as negative regulators of axonal guidance, angiogenesis and tumor progression. It has been demonstrated previously that one SEMA3, SEMA3F, may have therapeutic potential in the treatment of cancer. When transfected with SEMA3F, the highly metastatic human melanoma cell line A375SM was found to exhibit a highly-encapsulated, avascular phenotype with limited metastasis. Members of SEMA3 are regulated on many levels, including proteolytic processing. SEMA3F, like other SEMA3, is expressed as a 100 kD proprotein that is seen to be processed in vitro and in vivo to 95 and 65 kD isoforms. This has been largely attributed to furin-like endoproteases on the basis of furin inhibition studies. However, currently available small chemical or peptide inhibitors against the family of subtilisin/kexin-type proprotein convertases (PCSK), to which furin belongs, do not have good selectivity between PCSKs. Cleavage of SEMA3 to 65 kD have been shown to have differing effects. SEMA3A loses its ability to repel sympathetic ganglia and SEMA3E reverses its phenotype from chemorepulsant to chemoattractant for developing vasculature following cleavage. In order to further develop therapeutic strategies based on SEMA3F, it is therefore critical to better understand the proteolytic regulation of this molecule. In this study, it is shown that digest of purified SEMA3F with purified recombinant human furin does not result in proteolytic cleavage and suggested that the cleavage of SEMA3F to a 65 kD isoform may be mediated by other members of the PCSK family.