Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

A series of phenanthrene and oxidized phenanthrene derivatives with typical substitution patterns, 1.31, 1.32, 2.30a-f, 3.1a,b and (-)-4.1 have been synthesized as liquid crystal cores by a combined Directed ortho Metalation (DoM), cross coupling and Directed Remote Metalation (DreM) strategy. The synthetic methodology employed allowed variation of the tail and core structures, for the preparation of a new smectic liquid crystal compound (1.32), a homologous series of 9,10-dihydrophenanthrene-9,10-diones (2.30a-f), a diastereomeric trans-9,10-dihydrophenanthrene-9,10-diol (1.31), two enantiomeric 9,10-dihydrophenanthrene-9,10-diones (R)-3.1a-b) and an enantiomeric 9,10-dihydro-9,10-dimethylphenanthrene-9,10-diol ((-)-4.1). Polarized microscopic and differential scanning calorimetric measurements suggest that 1.31 forms a large range of SmC phase, ca. 100 ºC, followed by a small range of nematic phase, ca. 10 ºC; the 2.30a-f series show similar mesogenic properties, but with the extension of the length of a side chain from six carbons to eleven carbons, the nematic phase has disappeared (in the cases of 2.30a-c). Although (R)-3.1a-b and (-)-4.1 are not liquid crystals, they show ferroelectric induction by doping, in the amount of ca. 5 mol%, into unchiral liquid crystal hosts PhB, DFT, PhP1, NCB76 and 2.30f. However, due to the detection limit of the instrument, i.e., ca. 0.5 nC/cm2, the spontaneous polarizations (Ps) induced could not be measured. / Thesis (Ph.D, Chemistry) -- Queen's University, 2009-08-17 15:52:54.612

A major hurdle facing in vitro protein characterization is obtaining soluble protein from targets that tend to aggregate and form insoluble inclusion bodies. Soluble protein is essential for any biophysical data collection and new methods are needed to approach this significant problem. Directed evolution can be used to discover mutations which lead to improved solubility using an appropriate screening method. Green fluorescent protein (GFP) has been shown to be an effective solubility reporter which can be used to screen for soluble protein variants. We have chosen three diverse enzymes as targets for improving protein solubility using this technique: arachidonate 5-lipoxygenase—an enzyme which converts fatty acids into leukotrienes, PhnG—an enzyme belonging to the bacterial carbon-phosphorus lyase pathway, and RebG—a glycosyltransferase. Error-prone PCR and DNA shuffling were used to generate libraries of mutants which were subsequently cloned into a GFP-fusion screening vector. From the evolution of 5LO and RebG, much was learned about the optimization of the protocols involved in this methodology, including valuable information about how to avoid common “false-positive” results in which fluorescent colonies arise while screening but do not represent an improvement of the target. Evolution of these two targets did not result in an improvement of solubility, however truncation strategies may still prove to be effective, and more work needs to be done in this area. Evolution of PhnG successfully produced one variant, named clone B6, which showed both an improvement in expression and folding over wild type PhnG. It was also discovered that GFPuv can act as an effective solubility enhancing fusion tag for PhnG. Prior to the current studies PhnG had not been effectively expressed and purified in E. coli , however purification and refolding of resolubilized inclusion bodies of the clone B6 PhnG-GFP fusion construct was shown to yield enough soluble protein for future crystallographic studies. / Thesis (Master, Chemistry) -- Queen's University, 2009-10-09 12:26:03.353

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Disruptive behavior disorders including Attention-Deficit/Hyperactivity Disorder (ADHD), Conduct Disorder (CD), and Oppositional Defiant Disorder (ODD), are listed among the most common reasons youth are referred for mental health services (Centers for Disease Control & Prevention [CDC], 2016b; Kazdin, Mazurick, Siegel, & 1994). Parent-Child Interaction Therapy (PCIT) is one intervention that has been found to reduce clinically significant levels of disruptive behavior. The purpose of the current study was to determine the form of change, typical change trajectory, and individual variation in change for disruptive behavior across the two phases of PCIT; the Child-Directed Interaction (CDI) and Parent-Directed Interaction (PDI) phases. In addition, the current study determined which child and caregiver characteristics were associated with variation in change across CDI and PDI. Participants included a total of 75 children in PCIT between the ages of 2 to 8 years. Children and their caregiver(s) attended PCIT weekly at a university-based, outpatient clinic. The Eyberg Child Behavior Inventory (ECBI) was completed at every treatment session to indicate the intensity of disruptive behavior. Child and caregiver characteristics including the caregiver and the child’s gender, the caregiver’s income and marital status, the caregiver’s relationship with the child, the number of caregivers in PCIT, the child’s primary diagnosis, and the child’s medication status, were obtained through medical record abstraction. Results indicated the form of change in disruptive behavior, as measured on the ECBI Intensity scale, was linear in CDI and curvilinear in PDI. The average trajectory indicated disruptive behavior decreased throughout PCIT treatment. The decrease in ECBI Intensity scores during CDI was statistically significant, as well as the variance in children’s ECBI Intensity scores at the beginning of PDI. Caregiver marital status significantly predicted the ECBI Intensity score, which was higher for the divorced or separated group at the first session of PDI than other groups. Caregiver type also significantly predicted the ECBI Intensity score. When the caregiver was a grandparent, the ECBI Intensity score was lowest at the first session of PDI. However, the change in the ECBI Intensity slope for the biological parent group was steeper in comparison when transitioning from CDI to PDI, and less steep throughout PDI, than the grandparent group. Number of caregivers also was a significant predictor, with more caregivers present in PCIT indicating a greater decrease in the ECBI Intensity score. Significant child characteristic predictors were diagnosis code and medication status. For a diagnosis of Other (e.g., Adjustment Disorder, Selective Mutism), the ECBI Intensity score at the intercept was less than the ODD; ADHD; and Other Specified/Unspecified Disruptive, Impulse-Control, and CD groups; and higher than the Autism Spectrum Disorder/Social Pragmatic Communication Disorder group. A medication status of combined (greater than one psychopharmacological medication prescribed) indicated a higher ECBI Intensity score at the intercept, in comparison to the other groups. There was also a steeper change in slope throughout PDI when the diagnosis was ADHD in comparison to the ASD/SCD group. Last, when the medication status was single (one psychopharmacological medication prescribed), the change in slope during CDI for the ECBI Intensity score was steeper than the combined medication group. In summary, findings indicated disruptive behavior decreased during PCIT. However, clinicians and families may expect a slight increase in disruptive behavior at the beginning of PDI, or to see a slower rate of change in behavior, before the rate of change eventually speeds up and disruptive behavior decreases. Clinicians may see differences in the rate of change during PCIT based on caregiver and child characteristics and should use this information to guide discussions with families in the future. Future research should be conducted to determine if results may be replicated across different participant groups. Future studies may also follow-up on the maintenance of treatment gains after completing PCIT based on differences in rate of change for various caregiver and child characteristics examined in the current study.

Thesis advisor: Kian L. Tan / General and efficient methods for the selective hydroformylation of allylic alcohols and amines utilizing a directing group approach. / Thesis (MS) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The ability to detect a tactile stimulus during movement is markedly decreased (e.g., tactile gating), yet it is unknown whether the stimulus influences motor output. In the present study, participants moved a mechanical slider as quickly and as accurately as possible to a target. Participants received low-level electrical stimulation on the index finger of the reaching limb at various offsets relative to movement initiation. Participants reported whether they perceived the tactile cue. It was hypothesized that the detection of the stimulus would be reduced and the stimulus would influence motor output. First, a typical time course and magnitude of sensory gating was found, supporting previous observations (e.g., Chapman & Beauchamp, 2006). Second, no influence of the stimulation on motor output was observed. It was concluded that the detection of tactile cues during a goal-directed reaching task is attenuated and this stimulation does not influence motor output.

The ability to detect a tactile stimulus during movement is markedly decreased (e.g., tactile gating), yet it is unknown whether the stimulus influences motor output. In the present study, participants moved a mechanical slider as quickly and as accurately as possible to a target. Participants received low-level electrical stimulation on the index finger of the reaching limb at various offsets relative to movement initiation. Participants reported whether they perceived the tactile cue. It was hypothesized that the detection of the stimulus would be reduced and the stimulus would influence motor output. First, a typical time course and magnitude of sensory gating was found, supporting previous observations (e.g., Chapman & Beauchamp, 2006). Second, no influence of the stimulation on motor output was observed. It was concluded that the detection of tactile cues during a goal-directed reaching task is attenuated and this stimulation does not influence motor output.

The genetic defects that results in TAL1 oncogene activation are commonly seen in leukemic cells of the patient with T-cell Acute Lymphoblastic Leukemia ( T-ALL ). The ectopic expression of TAL1 oncoprotein perturbs the development of T-cell, hence promotes the formation of leukemia. TAL1 gene encodes proteins with basic helix-loop-helix ( bHLH ) domain, a protein dimerization and DNA binding domain. In T-ALL cells, two Tal1 proteins, pp42(1-331 amino acids) and pp22(176-331 amino acids) are produced that both contain bHLH domain. Both proteins interact with immunoglobulin gene enhancer binding protein, E12/E47 to form DNA-binding heterodimers, that can bind to consensus E-box DNA sequence AACAGATGGT. Phosphorylation of S122 residue modulates the trans-activation potential of Tal1 protein. In addition, S172 is an inducible c-AMP dependent protein kinase (PKA) phosphorylation site in vivo. The phosphorylation of TAL1 S172 upon stimulation by forskolin can increase the DNA binding of E12-Tal1 heterodimer. We used site-directed mutagenesis to investigate the effect of S194,S224 mutation on the function of truncated Tal1 oncoprotein.Mutant Tal1 and E12 proteins expression plasmids were constructed and introduced into COS-1 cells by cotransfection. Tal1 and E12 protein expression in transfected cell were evaluated by Western blotting. The protein-DNA interaction were evaluated by electrophorectic mobility shift assay. The mutation of S194 and S224 of Tal1 protein all resulted in the loss of DNA-binding complex formation. This data indicated that these serine residues are essential for bHLH function. However, the phosphorylation status of these two residues in vivo, and what kinase is responsible for the phosphorylation of these residues, await further investigation.