The expansion of gene-specific trinucleotide repeats is responsible for a growing list of human disorders, including myotonic dystrophy type 1 (DM1). Repeat instability for most of these disorders, including DM1, is characterized by complex patterns of inherited and ongoing tissue-specific instability and pathogenesis. While the mechanistic basis behind the unique locus-specific instability of trinucleotide repeats is currently unknown, DNA metabolic processes are likely to play a role. My thesis involves investigating the contribution of DNA replication to the trinucleotide instability of myotonic dystrophy type 1. Herein I have designed an in vivo primate model system, based on the SV40 replication system, to assess the contribution of DNA replication to DM1 repeat instability. This system allows the assessment, under controlled conditions, and manipulation of variables that may affect replication-associated repeat instability, under a primate cellular system. Using the SV40 model system, I not only confirmed previous observations that repeat length and replication direction affect repeat instability, but also for the first time determined that the location of the replication origin relative to the repeat tract plays an important role in repeat instability. This novel observation allowed for the development of a fork-shift model of repeat instability, in which cis-elements adjacent to the repeat tract affect replication, in turn altering the propensity for repeat instability. To further my study of DNA replication in DM1 repeat instability, I have mapped the origin of replication adjacent to the DM1 locus in human patient cells and the tissues of DM1 transgenic mice actively undergoing repeat instability. The position of the replication origins adjacent to the repeat tract at the DM1 locus places several known cis-elements, including CTCF binding sites, in a position to alter replication as predicted by the fork-shift model. My analysis of the CTCF sites showed them capable of altering replication and repeat instability at the DM1 locus. Taken together these results suggest that the placement of replication origins, repeat tracts and cis-elements, may mark trinucleotide repeat tracts, such as the DM1, for locus-, tissue- and development-specific replication-associated repeat instability.

<strong>Chapter 1</strong> introduces the concept of spin, how spins interact, and how the spin state in a radical pair can affect the outcome of a chemical reaction between the unpaired electrons. The computational methodology for simulating such radical pairs is also discussed. <strong>Chapter 2</strong> discusses anisotropy in the singlet recombination yield of a radical pair in a carotenoid-porphyrin-fullerene triad, containing many hyperfine couplings. The singlet yield was calculated as a function of the direction of an applied magnetic field, using symmetry in the molecule to reduce the size of the problem. The symmetry reduction was partially successful, however it was not possible to include all the hyperfine couplings in the molecule. <strong>Chapter 3</strong> introduces a radical pair located on a flavin ligand and a tryptophan residue in the protein cryptochrome, and discusses the spin-relaxation mechanism of singlet-triplet dephasing. Magnetic field effect curves, describing the formation of a secondary radical pair as a function of applied magnetic field, were found to be broader in longer-lived radical pairs, due to dephasing caused by spin-selective recombination to the singlet ground state. Additional singlet-triplet dephasing may occur due to hopping of one of the unpaired electrons, between a zone of strong exchange interaction and a zone of negligible exchange interaction, although this is an incomplete description of the spin-relaxation. <strong>Chapter 4</strong> discusses the effect of rotational tumbling on spin-relaxation in the flavin-tryptophan radical pair in cryptochrome. Simulations indicated that the resulting modulation of anisotropic hyperfine couplings contributed modestly to spinrelaxation during transient absorption measurements, but was insufficient to explain the lack of an experimental low-field effect, or to explain the width of the experimental magnetic field effect curves as a function of magnetic field strength. <strong>Chapter 5</strong> discusses magnetic field effects on the mutual annihilation of a pair of triplet excitons in tetracene and anthracene crystals. The experimental singlet recombination yield was found, for the first time, to be modulated as a function of the direction of a applied magnetic field as weak as 2 mT. Simulations indicated that this anisotropy arose due to the zero field splitting of the electronic state in each triplet exciton. The direction of the external magnetic field altered the singlet component of the eigenstates of the Hamiltonian, and therefore altered the timeaverage of the singlet probability of a triplet exciton pair. This is different to the already established mechanism under a strong magnetic field, where the anisotropy arises from level crossings of eigenstates.

abstract: Pedagogical content knowledge (PCK) has been described as the knowledge teachers' use in the process of designing and implementing lessons to a particular group of students. This includes the most effective representations that make the content understandable to students, together with the preconceptions and misconceptions that students hold. For chemistry, students have been found to have difficulty with the discipline due to its reliance upon three levels of representation called the triplet: the macro, the submicro, and the symbolic. This study examines eight beginning chemistry teachers' depiction of the chemistry content through the triplet relationship and modifications as a result of considering students' understanding across the teacher's first three years in the classroom. The data collected included classroom observations, interviews, and artifacts for the purpose of triangulation. The analysis of the data revealed that beginning chemistry teachers utilized the abstract components, submicro and symbolic, primarily in the first year. However, the teachers began to engage more macro representations over time building a more developed instructional repertoire. Additionally, teachers' developed an awareness of and responded to their students' understanding of learning atomic structure during the second and third year teaching. The results of this study call for preservice and induction programs to help novice chemistry teachers build a beginning repertoire that focuses on the triplet relationship. In so doing, the teachers enter the classroom with a repertoire that allows them to address the needs of their students. Finally, the study suggests that the triplet relationship framework should be revisited to include an additional component that frames learning to account for socioscientific issues and historical contributions. / Dissertation/Thesis / Ph.D. Curriculum and Instruction 2012

xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the protein MBNL1 (M[barbelow]uscleb[barbelow]lin[barbelow]d-l[barbelow]ike-1) is sequestered to the expanded CUG or CCUG RNAs. MBNL1 regulates the alternative splicing of a variety of pre-mRNAs and its mis-localization results in mis-splicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. I initially demonstrated that MBNL1 can bind short structured CUG and CCUG repeats with high affinity and specificity in vitro . Next, I was able to determine and articulate the first structure of a binding site of MBNL1 in an endogenous pre-mRNA that it regulates. I found that MBNL1 binds a stem-loop in the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. I determined that MBNL1 regulated exon 5 by directly competing with the essential splicing factor U2AF65 for binding upstream of exon 5. When U2AF65 is prevented from binding, factors in the spliceosome can no longer be recruited and the following exon is skipped. Furthermore, I found that MBNL1 and U2AF65 compete by binding mutually exclusive RNA structures. I also characterized a potential therapeutic approach for DM. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. Using a targeted screen of small molecules known to bind structured nucleic acids, I identified the small molecule pentamidine as a compound that disrupted MBNL1 binding to CUG repeats in vitro . I showed in cell culture that pentamidine was able to reverse the mis-splicing of two pre-mRNAs affected in DM. Pentamidine also significantly reduced the formation of RNA foci in tissue culture cells, which are characteristic of DM. MBNL1 was released from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of two pre-mRNAs in mice expressing expanded CUG repeats. This dissertation includes three previously published co-authored publications. / Committee in charge: Kenneth Prehoda, Chairperson, Chemistry; J. Andrew Berglund, Advisor, Chemistry; Victoria DeRose, Member, Chemistry; Peter von Hippel, Member, Chemistry; Alice Barkan, Outside Member, Biology

abstract: The first chapter reviews three decades of artificial photosynthetic research conducted by the A. Moore, T. Moore, and D. Gust research group. Several carotenoid (Car) and tetrapyrrole containing molecules were synthesized and investigated for excitation energy transfer (EET), photoregulation, and photoprotective functions. These artificial photosynthetic compounds mimicked known processes and investigated proposed mechanisms in natural systems. This research leads to a greater understanding of photosynthesis and design concepts for organic based solar energy conversion devices. The second and third chapters analyze the triplet energy transfer in carotenoid containing dyads. Transient absorption, time-resolved FTIR and resonance Raman spectra revealed that in a 4-amide linked carotenophthalocyanine dyads the Car triplet state is shared across the larger conjugated system, which is similar to protein complexes in oxygenic photosynthetic organisms. In a carotenopurpurin dyad (CarPur) a methylene ester covalent bond prevents the purpurin (Pur) from influencing the Car triplet based on the transient absorption, time-resolved FTIR and resonance Raman spectra. Thus CarPur resembles the antenna proteins from anoxygenic photosynthetic bacteria. Additional examples of carotenoporphyrin dyads further demonstrates the need for orbital overlap for ultrafast triplet energy transfer and the formations of possible intramolecular charge transfer state. The fourth chapter studies a 4-amino phenyl carotenophthalocyanine and its model compounds using high temporal resolution transient absorption spectroscopy techniques. EET from the Car second excited (S2) state to the phthalocyanine (Pc) was determined to be 37% and a coupled hot ground state (S*)/Pc excited state spectrum was observed. Excitation of the tetrapyrrole portion of the dyad did not yield any kinetic differences, but there was an S* signal during the excited states of the dyad. This demonstrates the EET and photoregulating properties of this artificial photosynthetic compound are similar to those of natural photosynthesis. The last chapter covers the synthesis of silicon Pc (SiPc) dyes and the methods for attaching them to gold nanoparticles and flat gold surfaces. SiPc attached to patterned gold surfaces had unperturbed fluorescence, however the selectivity for the gold was low, so alternative materials are under investigation to improve the dye's selectivity for the gold surface. / Dissertation/Thesis / Ph.D. Chemistry 2014

A presente tese descreve o estudo cinético e mecanístico da desativação do estado singlete- e triplete-excitado de flavinas por esteróis (colesterol e ergosterol), vitamina D, coenzima Q10, vitamina K, ácidos graxos e metabólitos da cafeína. Através da análise de Stern-Volmer da supressão de fluorescência da riboflavina pelo colesterol, ergosterol, vitamina D, vitamina K, e pela coenzima Q10 observa-se que o estado singlete excitado da riboflavina é desativado com constante de velocidade superior ao limite da difusão. No entanto, a presença de colesterol, ergosterol, vitamina D, vitamina K e coenzima Q10 não afeta o tempo de vida do estado singlete excitado da riboflavina como demonstrado por experimentos de contagem de fótons resolvido no tempo sugerindo a formação de um complexo precursor [riboflavina...substrato]. O complexo 1:1 formado entre a riboflavina e a vitamina D apresenta Ka = 4 x 104 &plusmn; 3 mol&sdot;L-1 a 25 0C com &Delta;H0 = -36 &plusmn; 7 kJ&sdot;mol-1 e &Delta;S0 = -5 &plusmn; 3 J&sdot;mol-1 &sdot;K-1. O complexo 1:1 entre a riboflavina e a vitamina K (vit K) e a riboflavina com a coenzima Q10 (CoQ10) apresentam Ka = 1 x 103 &plusmn; 1 mol&sdot;L-1 com &Delta;H0 = -110 &plusmn; 22 kJ&sdot;mol-1 e &Delta;S0 = 51 &plusmn; 9 J &sdot;mol-1&sdot;K-1 para a vit K e Ka = 4 x 102 &plusmn; 1 mol&sdot;L-1, &Delta;H0 = -120 &plusmn; 27 kJ &sdot;mol-1 e &Delta;S0 = 41 &plusmn; 7 J &sdot;mol-1&sdot;K-1 para a CoQ10 a 25 0C. Para a desativação do estado triplete da riboflavina, foram obtidas constantes bimoleculares de velocidade variando de 1,4 x 108 L &sdot;mol-1&sdot;s-1 (vit D) a 1,4 x 109 L &sdot;mol-1&sdot;s-1 (CoQ10). Observa-se supressão da emissão de fluorescência da riboflavina na presença de metabolitos da cafeína, no entanto, sem alterar o tempo de vida do estado singlete excitado da riboflavina sugerindo a formação de um complexo precursor [riboflavina...substrato]. O complexo formado entre a riboflavina e os metabólitos da cafeína apresentam Ka = 295 &plusmn; 1 mol&sdot;L-1 com &Delta;H0 = -45 &plusmn; 8 kJ&sdot;mol-1 e &Delta;S0 = 12 &plusmn; 1 J&sdot;mol-1&sdot;K-1 para o ácido 1,7-dimetil úrico, Ka = 289 &plusmn; 1 mol&sdot;L-1 com &Delta;H0 = -38 &plusmn; 5 kJ &sdot;mol-1 e &Delta;S0 = 9 &plusmn; 2 J &sdot;mol-1&sdot;K-1 para o ácido 1-metil úrico e Ka = 275 &plusmn; 1 mol&sdot;L-1 com &Delta;H0 = 16 &plusmn; 3 kJ &sdot;mol-1 e &Delta;S0 = 6 &plusmn; 1J &sdot;mol-1&sdot;K-1 para a 1,7-dimetilxantina a 25 0C. Para a desativação do estado triplete da riboflavina, foram obtidas constantes de velocidade de 3kq = 4,2 x 108 L.mol-1.s-1 para a 1,7-dimetilxantina, 3kq = 1,0 x 108 L.mol-1.s-1 para o ácido 1,7-dimetil úrico e 3kq = 1,4 x 108 L.mol-1.s-1 para o ácido 1-metil úrico. Os ésteres metílicos (oleato de metila, ácido linoléico conjugado (CLA), linoleato de metila, linolenato de metila, araquidonato de metila, eicosapentanoato de metila, e docosahexanoato de metila) não desativam o estado singlete-excitado da riboflavina. Entretanto, os ésteres metílicos se mostraram reativos frente ao estado triplete da riboflavina com constantes de velocidades 3kq variando de 8,4 x 105 a 3,3 x 107 L&sdot;smol-1 &sdot;s-1 com uma dependência linear do número de hidrogênios bis-alílicos com excessão do CLA. / The present thesis describes the kinetic and mechanistic studies of flavin singlet- and triplet-excited states deactivation by sterols (ergosterol and cholesterol), vitamin D, coenzyme Q10, vitamin K, fatty acids, and caffeine metabolites. From the Stern-Volmer analysis of the riboflavin fluorescence quenching by cholesterol, ergosterol, vitamin D, vitamin K and coenzyme Q10, it is noted that the singlet-excited state of riboflavin is deactivated with a rate constant exceeding the diffusion limit. However, the presence of cholesterol, ergosterol, vitamin D, vitamin K, coenzyme Q10 did not affect the lifetime of singlet-excited riboflavin as probed by single photon counting experiments suggesting the formation of a ground-state precursor complex [riboflavin ...substrate]. The 1:1 complex formed between riboflavin and vitamin D showed Ka = 4 x 104 &plusmn; 3 mol&sdot;L-1 a 25 0C with &Delta;H0 = -36 &plusmn; 7 kJ&sdot;mol-1 and &Delta;S0 = -5 &plusmn; 3 J&sdot;mol-1 &sdot;K-1. The 1:1 complex formed between riboflavin and vitamin K (vit K) and riboflavin with coenzyme Q10 (CoQ10) showed Ka = 1 x 103 &plusmn; 1 mol&sdot;L-1 with &Delta;H0 = -110 &plusmn; 22 kJ&sdot;mol-1 and &Delta;S0 = 51 &plusmn; 9 J &sdot;mol-1&sdot;K-1 for vit K e Ka =4 x 102 &plusmn; 1 mol&sdot;L-1, &Delta;H0 = -120 &plusmn; 27 kJ &sdot;mol-1 and &Delta;S0 = 41 &plusmn; 7 J &sdot;mol-1&sdot;K-1 for CoQ10 a 25 0C. For the deactivation of triplet riboflavin, bimolecular rate constants were found to vary from 1,4 x 108 L &sdot;mol-1&sdot;s-1 (vit D) a 1,4 x 109 L &sdot;mol-1&sdot;s-1 (CoQ10). The caffeine metabolites quench fluorescence emission of riboflavin however, without affecting the lifetime of the singlet-excited state, suggesting the formation of a ground state precursor complex [riboflavin ... substrate]. The complex formed between riboflavin and caffeine metabolites showed Ka = 295 &plusmn; 1 mol&sdot;L-1 with &Delta;H0 = -45 &plusmn; 8 kJ&sdot;mol-1 and &Delta;S0 = 12 &plusmn; 1 J&sdot;mol-1&sdot;K-1for 1,7-dimetyl uric acid, Ka = 289 &plusmn; 1 mol&sdot;L-1 with &Delta;H0 = -38 &plusmn; 5 kJ &sdot;mol-1 and &Delta;S0 = 9 &plusmn; 2 J &sdot;mol-1&sdot;K-1 for 1-methyl uric acid and Ka = 275 &plusmn; 1 mol&sdot;L-1 with &Delta;H0 = 16 &plusmn; 3 kJ &sdot;mol-1 and &Delta;S0 = 6 &plusmn; 1J &sdot;mol-1&sdot;K-1 for 1,7-dimethylxanthine at 25 0C. For the deactivation of triplet riboflavin, rate constant were obtained with 3kq = 4,2 x 108 L.mol-1.s-1 para a 1,7-dimethylxanthine, 3kq = 1,0 x 108 L.mol-1.s-1 for 1,7-dimethyl uric acid, and 3kq = 1,4 x 108 L.mol-1.s-1 for 1-methyl uric acid. The methyl esters (methyl oleate, conjugated linoleic acid (CLA), methyl linoleate, methyl linoleate, methyl arachidonate, methyl eicosapentanoate, and methyl docosahexanoate) did not quench the singlet-excited riboflavin. However, the methyl esters were shown to be reactive towards triplet riboflavin with rate constants ranging from 8,4 x 105 a 3,3 x 107 L&sdot;mol-1 &sdot;s-1and depending linearly with the number of bis-allylic hydrogens with exception to CLA.

The photosensitized processes significantly enhance photolysis of various chemicals in the aqueous system with dissolved organic matter (DOM) as sensitizer. The excitation of chromophores on the DOM molecule further generates reactive species as triplet states DOM, singlet oxygen, hydroxyl radical, carbonate radical etc. We investigated the photosensitization properties of Beaufort Fulvic Acid, Suwannee River Fulvic Acid, South Platte River Fulvic Acid, and Jeddah wastewater treatment plant effluent organic matter with a sunlight simulator. DOM photochemical properties were characterized by observing their performances in 3DOM*, singlet oxygen, hydroxyl radical production with indirect probing protocols. Sensitized degradation of 0.1 μM and 0.02 μM 2, 4, 6- Trimethylphenol exhibited higher pseudo-first-order rate constant than that of 10 μM. Pre-irradiated DOMs were found to be depressed in photochemical properties. Photolysis of 5 different contaminants: ibuprofen, bisphenol A, acetaminophen, cimetidine, and caffeine were found to be enhanced in the presence of sensitizers. The possible reaction pathways were revealed. Long time irradiance induced change in contaminants degradation kinetics in some DOM solutions, which was proposed to be due to the irradiation initiated indirect transformation of DOMs. Key Words: Photolysis Dissolved Organic Matter, Triplet State DOM, Singlet Oxygen, Hydroxyl Radical, Contaminants Degradation.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21554号 / 理博第4461号 / 新制||理||1640(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 石田 憲二, 教授 前野 悦輝, 教授 松田 祐司 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21555号 / 理博第4462号 / 新制||理||1640(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 前野 悦輝, 教授 石田 憲二, 教授 寺嶋 孝仁 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22876号 / 理博第4642号 / 新制||理||1667(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺西 利治, 教授 島川 祐一, 教授 長谷川 健 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM