Thesis (M.S.)--University of Akron, Dept. of Polymer Science, 2008. / "December, 2008." Title from electronic thesis title page (viewed 01/13/2010) Advisor, Judit E. Puskas; Faculty Reader, Gary R. Hamed; Department Chair, Ali Dhinojwala; Dean of the College, Stephen Z. D. Cheng; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

by Shinichi Watanabe. / Vita. / Thesis (M.S.) - Florida State University. / Bibliography: leaves 87-96.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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This thesis is divided into two parts. Part 1 covers the biosynthesis of the fungal metabolite tenellin, and Part 2 the metabolism of β-aminoisobutyrate m Streptomyces sp. Tenellin is a yellow pigment of the fungus Beamaria bassiana. It is of mixed biosynthetic origin, being derived from a polyketide moiety and the amino acid L-phenylalanine. The timing of the C-methylations of the polyketide chain is discussed in Chapter 2, which describes attempts to incorporate deuterium labelled partially assembled putative intermediates into the polyketide. The biosynthesis of the pyridone ring of tenellin requkes the condensation of the polyketide moiety with a rearranged phenylpropanoid unit derived from phenylalanine. The nature of this intriguing intramolecular rearrangement is discussed in Chapters 3 and 4. A phenylalanine derived tetramic acid, proposed as an intermediate in the biosynthesis, has been synthesised, and used in biosynthetic investigations. The results of these investigations and the subsequent identification of tyrosine as a closer precursor to tenellin argue against its intermediacy. The failure of [2-(^13)C(^2)H(^15)N]-phenylalanine to become incorporated intact suggests a transamination process for phenylalanine / tyrosine prior to incorporation. Preliminary investigations suggest para-hydroxy phenyllactate may be die substrate for the rearranging enzyme and a more direct precursor to tenellin. β-Aminoisobutyrate, the end product of reductive thymine catabolism, contributes to both the propionate and butyrate pools in Streptomyces sp. The pathway of incorporation into the isobutyrate / butyrate pool has been investigated, and confirmed to be the reverse of that known to occur in L-valine metabolism. A mutant strain of Streptomyces avemitilis, unable to produce isobutyrate, was used due to low level incorporations into the branched-chain fatty acids. This work was carried out in collaboration with Dr. Hamish McArthur, Pfizer Central Research Division, Groton, USA, and Dr. Kevin Reynolds, Department of Pharmaceutical Science, University of Maryland.

Ultraviolet irradiation of DNA induces the formation of a number of mutagenic lesions. The most prolific of these is the cis-syn thymine dimer (formed maximally at 260 nm) and this has been implicated in the reaction pathways that lead to ultraviolet-induced carcinogenesis. In order that the molecular events underlying these neoplastic events be understood, it is imperative that the thymine dimers formed in ultraviolet-irradiated thymine containing systems be quantitated. In this laboratory, dimer quantitation is performed using reverse phase high performance liquid chromatography (HPLC) with ultraviolet (DV) detection and the data obtained has allowed a kinetic mechanism for lesion formation to be proposed. Such studies have used in vitro thymine containing substrates (aqueous thymine, thymidine, thymidylyl-3',5'thymidine, calf thymus DNA and pUC19 plasmid DNA) to generate the thymine dimer using DV irradiation. with the planned extension of this research to in vivo cellular systems (where DNA and hence thymine concentrations are intrinsically less than those of in vitro systems), a more sensitive technique for thymine dimer quantitation is required. An immunological approach to providing this technique was chosen. Here, DV-irradiated DNA was injected into rabbits whose immune system mounted a ' response (i.e. antibody production) to the DV-DNA antigen. Blood was drawn from the rabbits at regular intervals to obtain the antibodies. The technique of immunoblotting was chosen and developed to allow detection of the thymine dimer antigen. This involved the reaction between the UV-DNA antigen, the primary antibody (generated by the rabbit) and a secondary antibody conjugated to an enzyme, all of which were immobilized on a commercially available membrane system. Detection and quantitation of the immune complex immobilized on the membrane was performed using the technique of enhanced chemiluminescence. Upon addition of a chemiluminescent substrate (luminol) to the immune complex, the horseradish peroxidase enzyme catalysed the reaction of luminol, with one of the products being light of 425 nm to 430 nm. This light impinged on a luminescence film which was developed and printed using standard photographic techniques. The use of dilutions of the primary antibody in the immunoblotting protocol with enhanced chemiluminescent detection, allowed correlations of antibody dilutions with UV-DNA antigen to be made. This immunoblotting technique with enhanced chemiluminescent detection has been used successfully in detecting thymine dimer lesion formation at levels currently above the detection limit of the HPLC. It has also been used successfully in detecting and quantitating thymine dimers at levels undetectable by the HPLC. To this end it has proved to be 4000 to 8000 times more sensitive than the chromatographic technique. Any immunological technique requires that the antibody of interest be purified and characterized. Here, purification of the crude serum was performed using the classical technique of ammonium sulphate precipitation of proteins. As an alternative technique, affinity chromatography was performed on the crude serum using a Memsep 1000 affinity chromatography cartridge attached to a preparative HPLC system. Chromatographic data illustrating this purification are given. Characterization of the DV-DNA antigen was performed by considering the specificity of the antibody response in the laboratory animal. Support for the kinetic mechanisms previously proposed for pyrimidine dimer formation in DNA is also given in this work. Calf thymus DNA was irradiated and dimer yields obtained by immunoblotting. These were used in the computer programme CAKE together with the previously determined rate constants to determine simulated dimer yields. A good agreement between experimental and simulated data indicated the validity of the mechanism at a DNA concentration of 0.025 mg/ml. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.

Undergraduate honors paper--Mount Holyoke College, 2007. Program in Biochemistry. / Includes bibliographical references (leaves 107-108).

Clustered damage to DNA is a signature mark of radiation-induced damage, which involves damage to the nucleobases and/or DNA backbone. Double-strand breaks created by damaging agents are detrimental to cell survival leading to chromosomal translocations. Normal cells employ Non-homologous end-joining because of its faster kinetics, to suppress chromosomal translocations. However, the presence of complex DNA ends constitutes a significant challenge to NHEJ. Location of Thymine glycol (Tg) at DSB ends was a potential hindrance to end joining. The substrate with Tg at the third position (Tg3) from the DSB joined better than when present at the fifth position (Tg5). However, hNTH1 assay showed Tg5 to be a better substrate than Tg3 for BER, potentially explaining the increased Tg removal and decreased end joining of Tg5 in extracts. Nonetheless, there appeared to be no preference in the susceptibility of 5’-Tg substrates with Tg at the second and third positions from DSB ends. Polynucleotide kinase phosphatase is crucial in restoring the 3′ hydroxyl, and 5′ phosphate ends at strand breaks. No other enzyme is known to possess PNKP’s activity in mammalian cells at DSBs. Experiments done with PNKP knockout cells have shown some activity similar to PNKP, which appeared to be a part of NHEJ and was not pharmacologically inhibited by PNKP inhibitor. Additionally, core NHEJ factors XRCC4 and XLF influenced the activities of PNKP. Overall, these experiments suggest that Tg repair is dependent on the position from DSB and an alternative enzyme processes 3′- PO, and 5′-OH ends.