α-Hemolysin (αHL) is a biological nanopore, which is currently under investigation for implementation into a new method for DNA sequencing. It has been established that αHL is capable of discriminating the canonical bases (adenine, cytosine, guanine and thymine) when they are immobilised within a pore by means of a biotin•streptavidin complex. Work in this thesis develops this procedure for the discrimination of these standard nucleobases from the the epigenetic modifications of cytosine – 5-methylcytosine (5mC) and 5 hydroxymethylcytosine (hmC). Strategies to selectively modify and detect the modified bases are also explored. Introduction. DNA sequencing strategies from the initial methods employed by Sanger up to current techniques utilised for the sequencing of entire human genomes, are reviewed. The initial discoveries of the epigenetic modifications 5-methylcytosine and 5 hydroxymethylcytosine are discussed. The proposed effects these bases have in vivo and a number of methods for their detection are also covered. Finally, methods for the selective chemical modification of the DNA bases are reviewed. Results and Discussion. Initially, the biotin•strepatvidin immobilisation strategy is implemented for the discrimination of the epigenetically modified analogues of cytosine (5mC and hmC), without prior chemical modification. It is subsequently observed that an α-hemolysin mutant is capable of discriminating all six bases – adenine, cytosine, guanine, thymine, 5 methylcytosine and 5-hydroxymethylcytosine. A number of different chemical reactions are then investigated on the bis-TBS protected deoxynucleoside model system, for their ability to selectively modify one of the DNA bases. Two selective oxidation reactions are then further optimised for use on ssDNA. Finally, electrical recording experiments were used to investigate two selective chemical reactions, as well as the modification of the bases, by the cancer drug temozolomide.

Mitochondria are a major source of reactive oxygen species and are particularly vulnerable to oxidative stress. Mitochondrial dysfunction, methionine oxidation, and oxidative stress are thought to play a role in both the aging process and several neurodegenerative diseases. Two major classes of methionine sulfoxide reductases, designated MsrA and MsrB are enzymes that function to repair the enatiomers of methionine sulfoxide, met-(o)-S and met-(o)- R, respectively. This study focuses on the effect of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. The data show that loss of only one form of Msr in the mitochondria does not completely impair the function of the mitochondria. However, loss of both Msr proteins within the mitochondria leads to an increased ROS production and a diminished energy output of the mitochondria. These results support the hypothesis that Msr plays a key role in proper mitochondrial function. / by Jennifer Verriotto. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Oil from flaxseed (Linum usitatisssimum L.) contains hydrophobic cyclic peptides or cyclolinopeptides (CLs) comprising eight or nine amino acids. These bioactive compounds have potential therapeutic applications and may be used as scaffolds for increased utility. Two steps were undertaken to increase the potential utility of these compounds. Initially multigram quantities of flax CLs were highly enriched from flax oil. Subsequently new synthetic procedures were developed for modification of the CLs through the methionine group (Met). Finally, the utility of the modified CLs was tested in a number of applications. CLs were recovered from a crude oil extract that contain five CLs (CLA, CLC, CLE, CLJ and CLK). Oxidation of this mixture reduced the complexity of the mix to just three CLA, CLJ and CLK. CLJ and CLK were enriched then characterized by NMR and MS-MS methods. CLs containing methionine sulfoxide groups (Mso), CLC and CLE were isolated from crude mixture then selectively reduced to afford Met containing analogs: CLB and CLE'. The Met of modified CLs was used as a point for attachment of tags and couplers for various applications. Cyclic peptide modification through Met groups has not been reported previously. Synthetic methods were devised to introduce activating functional groups such as -CN, -COOH, -OH and -NH2 to the sulfur atom of Met. The modified CL conjugates were characterized using spectrometric techniques including 1D and 2D NMR spectrometry, as well as mass spectrometry. After activation the CLs were covalently linked to molecules or materials of interest including fluorescence tags (coumarin), affinity chromatography media and bovine serum albumin (BSA) for production of polyclonal antibodies. Fluorescence studies were performed in methanol, ethanol, dimethylformamide and acetonitrile to study the solvent effect. CLs attached to solid affinity matrix showed specific binding to apolipoprotein A1 after incubation with chicken serum. These CLs also act as hapten and have been used to couple BSA to produce polyclonal antibodies. Met modification was a satisfactory approach to produce a range of useful peptide products where more conventional methods of molecule attachment are not available.

<p>This project is predicated on the ability of the boranophosphate modification of siRNA to increase its therapeutic applicability for gene silencing in in vitro and in vivo systems. It has been shown that the boranophosphate (BH3-PO3) can overcome many of the limitations that are traditionally found when using RNAi, namely nuclease stability. The synthesis of siRNA modified with 5'-(alpha-P-borano)-nucleoside triphosphates (NTP) analogs alone and in combination with 2'-deoxy-2'-fluoro nucleoside triphosphate analogs were performed and optimized. It was found that normal RNA transcriptions showed the highest yield with higher NTP concentrations and shorter incubation times. Boranophosphate modified RNA and 2'F/borano modified RNA transcription yield was optimal at lower NTP concentrations and extended incubations. The boranophosphate NTPs and RNA were characterized with high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, indicating successful synthesis of NTPalphaB and 2'F NTPs. PAGE and mass spectrometry analysis were performed to ensure full-length transcription of the modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical properties of the modified homoduplex and heteroduplex siRNA. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), nuclease stability assays, and assessment of the lipophilicity of the modified siRNA by determining partition coefficients. </p><p>Modification of siRNA with boranophosphate and 2'fluoro/borano modified NTPs appears to have caused the homoduplexes and heteroduplexes to adopt a more B form-like helix that had lower Tm compared to unmodified RNA. The stability of the siRNA transcript to enzymatic hydrolysis by Exonuclease T was on the order of 2'fluoro/borano> normal = boranophosphate. Boranophosphate modification increased the stability of the transcript to enzymatic hydrolysis by the endonuclease RNase A, compared to both normal and 2' fluoro modified siRNA. Overall, the 2' fluoro/borano modified siRNA showed the greatest biological stability. Modification of the siRNA with increasing percentages of boranophosphates resulted in increasing lipophilicity of the molecule up to 60-fold, compared to both normal and 2' fluoro RNA. </p><p>A method to site-specifically modify the boranophosphate siRNA using T4 RNA ligase was also investigated. Finally, the siRNA in this work was tested in several in vitro systems, yielding promising results for the usage of boranophosphate siRNA for use against human viruses and cancers. It was shown that in for in vitro systems for human papillomavirus gene expression (HeLa, SiHa, and W12E) and luciferase expression (B16F10 cells), boranophosphate modified siRNA can specifically downregulate gene expression, and in the case of human papillomavirus, can downregulate cell growth.</p> / Dissertation

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The system of mixed function oxygenase (MFO system) participates in significant roles in the metabolism of endogenous compounds and xenobiotics. This system contains cytochrome P450, NADPH:cytochrome P450 reductase, and also there are assigned NADH: cytochrome b5 reductase and cytochrome b5. It was proved that cytochrome b5 can stimulate or inhibit cytochrome P450 (CYP)-dependent reactions and even change the ratio of resulting metabolites. The mechanism of cytochrome b5 action has not been fully elucidated yet. Elucidation of protein-protein interactions in MFO system and determination of topology of this system could explain the mechanism of cyt b5 action. The covalent cross- linking technique is suitable method for identifying protein-protein interactions within the membrane. Cytochrome b5 contains 3 methionines and in 2 cases the methionines are localized in a short hydrophobic C-terminal membrane anchor. Interactions with cytochrome P450 in the membrane environment can be identified by substitution of two methionine for photoactivatable analogue of methionine (photo-methionine) and subsequent photoactivation. This work is focused on expression and isolation of photo-cytochrome b5 (photo- cyt b5), cytochrome b5 analogue with incorporated photo-methionine. Conditions for photo-methionine...

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18315号 / 農博第2040号 / 新制||農||1020(附属図書館) / 学位論文||H26||N4822(農学部図書室) / 31173 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 安達 修二, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM