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Mitochondrial diseases are a group of heterogenous disorders that share common cellular consequences resulting from mitochondrial dysfunction: (i) decreased ATP production; (ii) increased reliance on alternative anaerobic energy sources; and (iii) increased production of reactive oxygen species. Objective: We evaluated the effect of a combination (COMB) therapy comprising creatine monohydrate, coenzyme Q1 and lipoic acid to target the above mentioned consequences using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies. Results: Compared with placebo, the COMB therapy resulted in lower resting plasma lactate concentrations, lower urinary 8-isoprostane excretion and attenuated the decline of peak dorsiflexion strength in all patient groups. Improved body composition was only observed in patients in the MELAS group. Interpretation: These results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future therapies should be designed to target specific mitochondrial diseases to provide the greatest therapeutic benefits for those patients. In addition, future studies employing larger sample sizes in homogeneous groups of patients will be required to determine whether such combination therapies will influence function and quality of life. / Thesis / Master of Science (MS)

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Thesis advisor: Steven D. Bruner / Thesis advisor: Mary Roberts / The dioxygenase DpgC performs a key step in the biosynthesis of 3,5-dihydroxyphenylglycine (DPG), a nonproteogenic amino acid found in the vancomycin family of antibiotics. Remarkably, DpgC performs a 4-electron oxidation without the use of metals or cofactors. The tools of synthetic organic chemistry, enzymology and structural biology were used to study this enzyme. We have solved the first structure of an enzyme of this oxygenase class, in complex with a bound substrate mimic. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is located adjacent to the site of oxidation on the substrate. The use of a designed, synthetic substrate analog allowed us to gain unique insights into the chemistry of oxygen activation. We systematically probed the importance of active site residues by engineering conservative changes using site-directed mutagenesis. The kinetic parameters of these constructs imply that the phenolic hydroxyls of the substrate are of particular importance. These conclusions were verified by kinetic evaluation of synthetic substrate analogs. We have synthesized cyclopropyl substrate derivatives to probe the electron transfer step. The single electron oxidation should produce a radical species capable of opening the cycloproyl ring, thus providing a handle of detection. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.

Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.

Splines are piecewise polynomial functions of a given order of smoothness r. Given complex delta the set of splines of degree less than or equal to d forms a vector space and is denoted by Sr d(delta). For a simplicial complex delta, Strang conjectured a lower bound on the dimension of spline space Srd(delta) and it is known that the equality holds for sufficiently large d. It is called the dimension formula. In this dissertation, we approach the study of splines from the viewpoint of algebraic geometry. This dissertation follows the works of Lau and Stiller. They introduced the conformality conditions which lead to the machinery of sheaves and cohomology which provided a powerful type of generalization of linear algebra. First, we try to analyze effects in the dimensions of spline spaces when we remove or add certain faces in the given complex. We define the cofactor spaces and cofactor maps from the given complexes and use them to interpret the changes in the dimensions of spline spaces. Second, given polyhedral complex delta, we break it into two smaller complexes delta1 and delta2 which are usually easier to handle. We will find conditions for delta1 and delta2 which guarantee that the dimension formula holds for the original complex delta. Next, we use the previous splitting method on certain types of triangulations. We explain how to break the given triangulation and show what kind of simple complexes we end up with. Finally, we study the "2r+1" conjecture on a certain triangulation. The "2r+1" conjecture is that the dimension formula holds on any triangulation for d >/= 2r + 1. We know that the conjecture is sharp because the dimension formula fails on a certain triangulation for d = 2r, but we do not know if it holds on the same triangulation when d = 2r + 1. It is related to a Toeplitz matrix.