abstract: Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of energy from the chlorosome complex to the reaction center of the bacteria. The three-dimensional structure of the protein has been solved to a resolution of 2.0Å with the Rwork and Rfree values being 16.6% and 19.9% respectively. This new structure is compared to the FMO protein structures of Prosthecocholoris aestuarii 2K and Chlorobium tepidum. The early structures of FMO contained seven bacteriochlorophyll-a (BChl) molecules but the recent discovery that there is an eighth BChl molecule in Ptc. aestuarii 2K and Cbl. tepidum and now in Pld. phaeum requires that the energy transfer mechanism be reexamined. Simulated spectra are fitted to the experimental optical spectra to determine how the BChl molecules transfer energy through the protein. The inclusion of the eighth BChl molecule within these simulations may have an impact on how energy transfer through FMO can be described. In conclusion, a reliable method of purifying and crystallizing the SMNWT protein is detailed, the placement of the 8th BChl-a within the electron density and the implications on energy transfer within the FMO protein when the 8th BChl-a is included from the green sulfur bacteria Pld. phaeum is discussed. / Dissertation/Thesis / Ph.D. Biochemistry 2010

The use of ethionamide has been increasing in drug regiments due to greater incidence of multidrug resistance tuberculosis around the world. Ethionamide is metabolized into antimicrobial relevant compounds by different flavin-containing monooxygenase (FMO) enzymes including FMO1, FMO2, and FMO3. FMOs are found in various locations in the body including the intestine, kidney, liver, and lung. In humans, active functional FMO2*1 is found in approximately 50% of Sub-Saharan Africans and a truncated, inactive FMO2*2, is found in all Caucasians and Asians. Polymorphisms in human FMO2 were investigated by comparing differences in metabolism of ethionamide in wildtype mice relative to Fmo 1/2/4 null mice. All mice were capable of metabolizing ethionamide into ethionamide S-oxide and 2-ethyl-4-amidopyridine. Wildtype mice had higher plasma levels of metabolites than parent compound. In contrast, Fmo 1/2/4 null mice had higher plasma levels of parent compound than metabolites. In both mouse populations, maximum ethionamide concentration peaked at 2 hours post-exposure. Increased metabolism of ethionamide in wildtype mice may deplete glutathione pools and induce oxidative stress leading to greater toxicity and adverse drug effects. This murine model is used to demonstrate the polymorphic differences of FMO2 occurring in humans. Taking these differences into account, polymorphisms of drug metabolizing enzymes provide a basis for increasing specific and individualized drug treatment regiments in susceptible populations. / Graduation date: 2012

Video coding standards such as H.264 AVC (Advanced Video Coding) rely on predictive coding to achieve high compression efficiency. Predictive coding consists of predicting each frame using preceding frames. However, predictive coding incurs a cost when transmitting over unreliable networks: frames are no longer independent and the loss of data in one frame may affect future frames. In this thesis, we study the effectiveness of Flexible Macroblock Ordering (FMO) in mitigating the effect of errors on the decoded video and propose solutions to improve the error concealment on H.264 decoders. After introducing the subject matter, we present the H.264 profiles and briefly determine their intended applications. Then we describe FMO and justify its usefulness for transmission over lossy networks. More precisely, we study the cost in terms of overheads and the improvements it offers in visual quality for damaged video frames. The unavailability of FMO in most H.264 profiles leads us to design a lossless FMO removal scheme, which allows the playback of FMO-encoded video on non FMO-compliant decoders. Then, we describe the process of removing the FMO structure but also underline some limitations that prevent the application of the scheme. Finally, we assess the induced overheads and propose a model to predict these overheads when FMO Type 1 is employed. Eventually, we develop a new error concealment method to enhance video quality without relying on channel feedback. This method is shown to be superior to existing methods, including those from the JM reference software and can be applied to compensate for the limitations of the scheme proposed FMO-removal scheme. After introducing our new method, we evaluate its performance and compare it to some classical algorithms.

The design of chromophores targets materials with optoelectronic properties necessary for advanced applications. Organic materials possess properties which emerge from the collective impact of the constituent backbone and substituents as well as their connectivity (i.e. molecular architecture), necessitating the exploration of novel conjugated architectures. This thesis chronicles our examination of 1,4-distyryl-2,5-bis(arylethynyl)benzenes (cruciforms, XFs). Electronic substitution of this 'X-shaped' cross-conjugated scaffold tunes both the energy levels and the spatial distribution of the frontier molecular orbitals (FMOs) in XFs. The resulting fluorophores exhibit FMO separation, imbuing XFs with desirable properties for sensory applications. Using model analytes, we examine how the underlying FMO arrangement and the nature of analyte interaction elicit observable responses. These studies provide a foundation for future access of functional responsive ratiometric cores. This case study demonstrates the importance and unique potential of FMO-separated fluorophores.

Two chlorophylls of the FMO complex, the light-harvesting complex of the green sulfur bacteria, are modeled as two coupled qubits, each surrounded by one spin-bath simulating the environment. The dynamics of the system at a non-zero temperature provide exact analytical expressions for the transition probability and the coherence. It is shown that the decoherence-inducing interaction with the environment enhances the electronic energy transfer. Also the correlations in terms of entanglement and nonlocality are quantitatively studied, sensitively differing when introducing a decay term to resemble both chlorophylls being in their ground states. It is proved that nonlocality is a stronger form of correlation than entanglement.

This thesis is concerned with quantum properties of excitonic energy transport in nanostructures that are embedded in a noisy environment. Of principal interests are ways to exploit this environment to facilitate the transport of energetic excitations. The first research chapter deals with an extension to the 'standard' open quantum system picture, where the Hilbert space is split into three: system, environment, and a wider universe. This division is natural for many biological and artificial nanostructures. A new analytical method, based on a phase space representation of the density matrix, is developed for studying such division. The effects of the wider universe are shown to be captured by a simple correction of the environmental response function. The second research chapter addresses the question: when do second-order perturbative approaches to open quantum systems, which are intuitive and simple to compute, provide adequate accuracy? A simple analytical criterion is developed, and its validity is verified for the case of the much-studied FMO dynamics as well as the canonical spin-boson model. In the third research chapter, an intuitive model of a photocell is studied. The model comprises two light-absorbing molecules coupled to an idealised reaction centre, showing asymmetric dimers are capable of providing a significant enhancement of light-to-current conversion under ambient conditions. This is done by 'parking' the energy of an absorbed photon in a dark state which neither absorbs nor emits light. In the final research chapter, a basic model for what can be thought as a "quantum brachistochrone" problem is investigated. Exotic energy configurations are found to yield considerable enhancement to the exciton's transfer probability, due to similar mechanisms studied in the previous chapter.

Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities. Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement. Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen, v methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor. The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined. The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p&lt / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.

The flavin-containing monooxygenases (FMO / E.C.1.14.13.8) are microsomal NADPH and oxygen-dependent flavoprotein enzymes that catalyze the oxidation of a wide variety of xenobiotics, including drugs and environmental toxicants. Nucleophiles containing nitrogen, sulfur, phosphorus and selenium heteroatoms are the substrates of FMO. Bovine liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO. From 12 different bovine liver samples, microsomes were prepared and the average specific activity of bovine liver microsomal FMO was found to be 2.37 &amp / #61617 / 0.30 nmol/min/mg (Mean &amp / #61617 / SE, n=12). The rate of reaction was linear up to 0.5 mg of bovine liver microsomal protein. The maximum FMO enzyme activity was detected at 37 &amp / #61616 / C and at pH 8.0. Effects of detergents / Triton X-100 and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations (0.1%-1.0%). The apparent Vmax and Km values of bovine liver microsomal FMO for methimazole substrate were found as 1.23 nmol/min/mg and 0.11 mM, respectively. Thermostability of bovine liver microsomal FMO was studied at four different temperatures / 24 &amp / #61616 / C, 37 &amp / #61616 / C, 50 &amp / #61616 / C and 65 &amp / #61616 / C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 &amp / #61616 / C, 10 minutes at 50 &amp / #61616 / C and 6.5 hours at 37 &amp / #61616 / C. 68 % of the activity was still detectable at the end of 53 hours at 24 &amp / #61616 / C. Bovine liver microsomal activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 3.73 and 3.75 nmol NADPH oxidized/min/mg, respectively. Effects of two drug substrates, imipramine and chlorpromazine, on bovine liver microsomal FMO-catalyzed methimazole oxidation activity was also studied and found that they inhibit FMO activity at all concentrations studied. Modulation of bovine liver microsomal FMO activity was studied using three different heavy metal ions / Ni+2, Cd+2 and Hg+2. At all other concentrations studied for each heavy metal ion and at all substrate methimazole concentrations (0.1, 0.2, 0.5, 1.0 mM), FMO-catalyzed methimazole oxidation activity decreased compared to control activity. KI values for Ni+2, Cd+2 and Hg+2 were found to be 0.5 mM, 0.085 mM, 4.6 &amp / #61549 / M, respectively. From the Dixon plot, the pattern of inhibition for three heavy metal ions was observed to be noncompetitive.

<div> <div> <div> <p>The Effective Fragment Potential (EFP) is a quantum-mechanical based model potential for accurate calculations of non-covalent interactions between molecules. It can be coupled with ab initio methods in so-called QM/EFP models to explore the electronic properties of extended molecular systems by providing rigorous description of surrounding environments. The current EFP formulation is, however, not well suited for large-scale simulations due to its inherent limitation of representing effective fragments as rigid structures. The process of utilizing EFP method for the molecular systems with flexible degrees of freedom entails multiple sets of parameter calculations requiring intensive computational resources. This work presents development of the EFP method for describing flexible molecular systems, so-called Flexible EFP. To validate the applicability of the Flexible EFP method, extensive benchmark studies on the amino acid interactions, binding energies, and electronic properties of flavin chromophore of the cryptochrome protein have been demonstrated. In addition to methodological developments, excitonic properties of the Fenna-Matthews-Olson (FMO) photosynthetic pigment-protein complex are explored. In biological systems where intermolecular interactions span a broad range from non-polar to polar and ionic forces, EFP is superior to the classical force fields. In the present study, we demonstrate excellent performance of the QM/EFP model for predicting excitonic interactions and spectral characteristics of the FMO wildtype complex. We characterize the key factors for accurate modeling of electronic properties of bacteriochlrophyll a (BChl a) photosynthetic pigments and suggest a robust computational protocol that can be applied for modeling other photosynthetic systems. Developed computational procedures were also successfully utilized to elucidate photostability and triplet dynamics in the FMO complex and spectroscopic effects of single-point mutagenesis in FMO. A combination of polarizable EFP molecular dynamics and QM/EFP vibrational frequency calculations were also applied to understanding and interpreting structures and Raman spectroscopy of tert-butyl alcohol solutions. </p> </div> </div> </div>

Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.