Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

Protein pK(a)s and metalloprotein reduction potentials are studied with computational methodologies based on an ab initio quantum mechanics (QM) description of the protein and a linearized Poisson-Boltzmann Equation (LPBE) description of the solvent. The practical applicability of the QM/LPBE method is extended to proteins by using a QM description of the ionizable residue and a molecular mechanics (MM) description of the rest of the protein. This QM/MM/LPBE method is used to predict the pKa of Lys55 in the serine protease inhibitor turkey ovomucoid third domain (OMTKY3) and the prediction of 11.0 is in good agreement with the experimental value of 11.1. This is the first time a protein pKa value has been predicted with QM/MM methods. The QM/LPBE method is used to predict and interpret the pKa values of the five carboxyl residues (Asp7, Glu10, Glu19, Asp27, and Glu43) in OMTKY3. All the predicted pKa values are within 0.5 pH units of experiment, with a root mean square deviation of 0.31 pH units. We find that the decreased pKa values observed for some of the residues are primarily due to hydrogen bonds to the carboxyl oxygens. Hydrophobic effects are also shown to be important in raising the pKa. Interactions with charged residues are shown to have relatively little effect on the carboxyl pKa values in this protein, in general agreement with experiment. The relative Cu2+/Cu+ reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied with the QM/LPBE method. For the first time, the range and relative orderings of the reduction potentials are reproduced well compared to experimental values. The study suggests that the main interactions determing the relative reduction potentials of blue copper sites are located within 6 Å of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraints on the inner sphere ligand orientations.

No description available.

<p>This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (p<i>K</i>_a) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic <i>NE</i>-type (P = 37° ± 27°, Φ_m = 25° ± 18°) for 1′,2′-constrained nucleosides, and <i>N</i>-type (P = 19° ± 8°, Φ_m = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)₂, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).</p>

This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (pKa) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic NE-type (P = 37° ± 27°, Φm = 25° ± 18°) for 1′,2′-constrained nucleosides, and N-type (P = 19° ± 8°, Φm = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).

Coupled electron and proton transfer (CEPT) events are fundamental for many bioenergetic conversions that involve redox reactions. Understanding the details underlying CEPT processes will advance our knowledge of (1) how nature regulates energy conversion; (2) our strategies for achieving renewable energy sources; (3) how to cope with CEPT dysfunction diseases. Studies of the detailed mechanism(s) of CEPT in biological systems is challenging due to their complex nature. Consequently, controversies between the concerted and sequential mechanism of CEPT for many systems remain. This dissertation focuses on the bovine mitochondrial cytochrome bc1 complex. CEPT in the bc1 complex operates by a modified "Q-cycle"(1) and catalyzes electron transfer from ubiquinol (QH2), to cyt c via an iron sulfur cluster (ISC) and to the low potential hemes of cyt b, where it reduces ubiquinone (UQ). The electron transfer is coupled to the translocation of protons across the mitochondrial inner membrane, generating a proton gradient that drives ATP synthesis. Although the Q-cycle is widely accepted as the model that best describes how electrons and protons flow in bc1, detailed binding geometries at the Qo site (QH2 oxidation site) and Qi site (UQ reduction site) remain controversial. The binding geometries play critical roles in the thermodynamic and/or kinetic control of the reaction and protonatable amino acid side chains can participate in the proton transfer. The central focuses of this dissertation are molecular dynamics simulations of cofactor binding geometries near the Qo and Qi sites, calculations of the pKa values of ionizable amino acid side chains implicated in cofactor binding, especially the ISC-coordinated histidines, and implications for the proposed mechanism(s) of CEPT. For the first time, pKa values of the ISC-coordinated histidines are differentiated. The computed pKa values of 7.8±0.5 for His141 and 9.1±0.6 for His161 agree well with experiment (7.63±0.15 and 9.16±0.28). Thus, His161 should be protonated at physiological pH and cannot be the first proton acceptor in the QH2 oxidation. Water mediated hydrogen bonds between substrate models and the protein and water accessibility to the Qo and Qi sites were maintained in simulations, implying that water molecules are likely the proton donors and acceptors. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

This thesis consists of seven research publications concerning (i) pKa studies of nucleobases in model nucleotides to understand why RNA duplexes are more stable than DNA duplexes (Paper I), (ii) the role of Me(T)-π interactions in the relative stability of DNA-RNA heteroduplexes (Paper II), (iii) pKa measurements in nucleotides with different 2′-substituents (paper III), (iv) a conformation study of constrained sugars and a pKa study of 1-thyminyl to reveal effect of sugar constraints on the pKa of the nucleobase (paper IV), (v) NMR and MD studies of 1′, 2′-oxetane constrained thymidine incorporated Dickerson Drew dodecamer (paper V), (vi) the sequence dependent pKa perturbation of 9-guaninyl moeity in single stranded (ss) DNA and RNA (paper VI), (vii) the non identical chemical nature of internucleotidic phosphates in (ss) RNA using 31P NMR (paper VI), and an alkaline hydrolysis study of phosphodiesters in ssRNAs (paper VII). The architecture of DNA and RNA molecules is determined by (a) hydrogen bonding (b) base stacking (c) a variety of additional non-covalent interactions. In paper (I) we showed that A-U and G-C base pairings in RNA are more stable than A-T and G-C base pairings in DNA by 4.3 and 1 kJ mol-1 respectively. Me(T)-π interaction plays a dominant role in the relative stability of DNA-RNA duplexes (paper II). In paper III and IV, we have shown that 1′ , 2′- oxetane and azetidine rings have strong inductive effect on pyrimidine bases, and that the H2′-sugar proton can be the marker to understand the pseudoaromaticity of pyrimidine bases, as well as increasing constraints in sugar reducing the basicity of nucleobases. A 1′, 2′-oxetane locked thymidine (T) moiety deforms the local structure of Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2- investigated by High resolution NMR and MD study, as is discussed in the paper V. In papers VI and VII, we showed sequence context dependent pKa (N1) of 9-guaninyl perturbation in (ss) DNAs and RNAs and the non identical chemical nature of inter-nucleotidic phosphate groups in single stranded RNAs.

Polarized Raman spectroscopy has been used to measure the deuterium isotope effect, (delta)pK = pKD2O – pKH2O, for the second ionization constant of sulfuric acid in the temperature range of 25 °C to 200 °C at saturation pressure. Results for pK in light water agree with the literature within ± 0.034 pK units at alltemperatures under study, confirming the reliability of the method. The ionization constant of deuterated bisulfate, DSO4-, differs significantly from previous literature results at elevated temperatures. This results in an almost constant (delta)pK ≈ 0.425 ± 0.076 over the temperature range under study. Differences in (delta)pK values between the literature and current results can be attributed to the effect of dissolved silica from cell components. The new results are consistent with (delta)pK models that treat the temperature dependence of (delta)pK by considering differences in the zero-point energy of hydrogen bonds in the hydrated product and reactant species. The phosphate hydrolysis equilibrium was measured between the temperatures of 5 °C and 80 °C and the borate/boric acid equilibrium between the temperatures of 25 °C and 200 °C. The high alkalinity and temperatures experienced by these two systems had a significant impact on the glass dissolution and equilibrium. / Raman spectroscopy was used to measure the small differences in ionization constants for weak acids/bases as a function of temperature. / University of Guelph, Atomic Energy of Canada Limited (AECL), Bruce Power, University Network of Excellence in Nuclear Engineering (UNENE), National Sciences and Engineering Research Council of Canada (NSERC), Natural Resources Canada, Ontario Power Generation (OPG), Canada Foundation for Innovation

The air kerma area product PKA is an important quantity used by hospital physicists in quality assurance and optimization processes in diagnostic radiology and is recommended by national authorities for setting of diagnostic reference levels. PKA can be measured using a transmission ionization chamber (kerma area product (KAP) meter) mounted on the collimator housing. Its signal QKAP must be calibrated to give values of PKA. The objective of this thesis is to analyze the factors influencing the accuracy of the calibration coefficients k= PKA/QKAP and of reported PKA-values. Due to attenuation and scatter in the KAP-meter and presence of extra-focal radiation, values of PKA depend on the choice of integration area A and the distance of the reference plane from the focal spot yielding values of PKA that may differ by as much as 23% depending on this choice. The two extremes correspond to (1) PKA=PKA,o integrated over the exit surface of the KAP-meter resulting in geometry independent calibration coefficients and (2) PKA=PKA,Anom integrated over the nominal beam area in the patient entrance plane resulting in geometry dependent calibration coefficients. Three calibration methods are analysed. Method 1 aims at determine PKA,Anom, for clinical use at the patient entrance plane. At standard laboratories, the method is used to calibrate with respect to radiation incident on the KAP-meter. Problems with extra-focal and scattered radiation are then avoided resulting in calibration coefficients with low standard uncertainty (±1.5 %, coverage factor 2). Method 2 was designed in this work to approach determination of PKA,o using thermoluminescent detectors to monitor contributions from extra-focal radiation and account for the heel effect. The uncertainty in derived calibration coefficients was ± 3% (coverage factor 2). Method 3 uses a Master KAP-meter calibrated at a standard laboratory for incident radiation to calibrate clinical KAP-meters. It has potential to become the standard method in the future replacing the tedious method 2 for calibrations aiming at determination of PKA,o. Commercially available KAP-meters use conducting layers of indium oxide causing a strong energy dependence of their calibration coefficients. This dependence is investigated using Monte Carlo simulations and measurements. It may introduce substantial uncertainties in reported PKA– values since calibration coefficients as obtained from standard laboratories are often available only at one filtration (2.5 mm Al) as function of tube voltage or HVL. This is not sufficient since higher filtrations are commonly used in practice, including filters of Cu. In extreme cases, calibration coefficients for the same value of HVL but using different tube voltages and filtrations can deviate by as much as 30%. If standardised calibration methods are not used and choice of calibration coefficients not carefully chosen with respect to beam quality, the total uncertainty in reported PKA–values may be as large as 40-45%. Conversion of PKA-values to risk related quantities is briefly discussed. The large energy dependence of the conversion coefficients, ε/PKA, for determination of energy imparted,ε, to the patient reduces to a lower energy dependence of calibration coefficients CQ,ε = ε/QKAP for determination of ε from the KAP-meter signal.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.