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<p>The two lipid kinases, diacylglycerol kinase (DGK) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), are vital players of the phosphatidylinositol cycle. DGK regulates the intracellular balance between two important lipid signaling molecules, diacylglycerol and phosphatidic acid. PIP5K produces another key signal messenger, phosphatidylinositol 4,5-bisphosphate. We studied several fundamental aspects of DGK and PIP5K properties. We investigated the topology of the hydrophobic segment of FLAG-tagged DGK epsilon, and showed that a single amino acid mutation P32A caused the hydrophobic segment to favor a transmembrane orientation. We demonstrated that DGKε is localized in both the plasma membrane and endoplasmic reticulum. Our work helped to better elucidate the substrate specificity of DGKε and PIP5K isoforms, and it lead us to discover the motif that is common for several enzymes that exhibit specificity for substrates containing polyunsaturated fatty acids. We studied the organ distribution of murine DGK isoforms, and also expanded our knowledge of DGK expression in diabetic animals, showing that the expression profiles of several DGK isoforms are altered in adipocytes isolated from diabetic mice. Moreover, DGK expression profiles change dramatically during adipocyte differentiation. Taken together, our findings contribute to the growing knowledge about two enzymes, DGK and PIP5K, by providing the fundamental information about the structural and functional properties of these lipid kinases. Both PIP5K and DGK enzymes have a strong potential for use as drug targets. Although at present their clinical importance has not been completely assessed, we believe that their significance as drug targets will be recognized in the nearest future. <strong></strong></p> / Doctor of Philosophy (PhD)

The purpose of this study was to examine the effect of encoding specificity on learning in a multimedia environment. Based upon the theory of encoding specificity there should be a relationship between the modality for which a learner encodes information into memory and the modality used to assess the learner's knowledge. Modality attributes for purposes of this study included visual (animation) and verbal information (narration and text). Two-hundred and fifteen students viewed a computer animation on lighting formation which was presented in one of three different modalities (animation with narration, animation with text, text only). Following the instruction students were assessed in one of three modalities (animation with narration, animation with text, text only) on recall and transfer. A 3 Encoding/Study x 3 Retrieval/Test (animation with narration, animation with text, text only) full-factorial post-test only design was used to assess the effects of matched and mismatched encoding and retrieval modalities in a multimedia environment. Encoding specificity suggests that there is an interaction between the conditions at encoding and retrieval such to say that the to-be-remembered item will not be as effective during retrieval unless the cue was specifically encoded at time of storage. Unfortunately, the present study did not find much to support the claim of encoding specificity based upon modality. The use of modality in both encoding and retrieval condition to support encoding specificity was found only in the AT-AT matched recall group versus the mismatched groups. Furthermore, significance was not found in any of the matched mismatched transfer conditions. / Ph. D.

Diversity of ectotrophic mycorrhizal (EM) fungi on out-planted seedlings of two oak species (Quercus rubra and Q. prinus) was estimated at two sites in mature mixed forests in the southern Appalachian mountains. Late – stage fungi were well represented. Total richness was 73 types, with 42 types having a frequency of only one. Thelephoroid / tomentelloid, russuloid, and cortinarioid groups were the richest. Dominant fungi included a putative Tuber sp. and Craterellus sp., and Laccaria cf laccata. Diversity was lower at a high elevation chestnut oak dominated site compared to a lower mesic cove – hardwood forest site. There was little evidence for fungal specificity to red oak versus white oak seedlings. We also compared EM fungus distributions on root systems of oak seedlings from samples taken in mid-July and early-September. The majority of EM types occurred only in the mid- or late-summer samples respectively. Dramatic shifts in mycobiont dominance were observed in relation to sample date, including increases in Cortinarius spp. richness, decreases in Thelephoraceae richness, and the disappearance of Amanita spp. types in the late- compared to mid-summer samples. A multi-stage model of seasonal EM dynamics is proposed, with implications for the niche expansion of associated phytobionts. In this model, generalistic mycobionts are most frequent and occur throughout the season. Other more specialized fungi show seasonal specificity. Relationships between species and communities of EM fungi and environmental parameters such as ericoid shrub abundance and edaphic characteristics were also examined. High diversity of EM fungi limited resolution of community level relationships given our sample sizes. Intraspecific variation in EM fungi with regard to microsite characteristics was also undetectable. No association between ericoid shrub dominance (Kalmia latifolia and Rhododendron maximum) and EM fungi was observed. We present a listing of EM fungus types with associated ranges of edaphic parameters and ericoid shrub abundance. The family Sebacinaceae is a basal hymenomycete lineage that includes members of the genera Tremellodendron and Sebacina. We present evidence suggesting the putative mycorrhizal status of two species of Tremellodendron. Tremellodendron appears to form both endophytic associations with achlorophyllous orchids and ectomycorrhizae with species of Quercus, Pinus and Tilia cordata. / Ph. D.

This study examined the effects of six weeks of velocity specific isokinetic training on peak torque (PT), and the estimated cross-sectional area of the upper arm (AG) in the trained. Thirty volunteers (M=15, F=15) were randomly assigned to an experimental, slow velocity group (S), 60 degrees-per-second (n=9; 25.4±..6.5yr), a fast velocity group (F), 450 degrees-per-second (n=ll, 23.7 ±..S.4yr), or control group (C) (n=10, 26 ± 3.2yr). One limb was randomly selected for isokinetic training (3 d/wk-elbow flexion) using a Biodex System 2 isokinetic dynamometer. The contralateral limb served as a control and as the basis for measurements measure of cross education (CE). Both experimental conditions (S) and (F) were assigned equal training workloads, calcu1ated from an isokinetic pre-test. Pre- and post-tests (PT) were recorded for both limbs at the training velocities of 60 and 450 degrees-per-second, as well as the velocity of 210 degrees-per-second. Pre and post-test (AG's) were measured on the training limb. The (S) condition was significantly different in strength gains from the control at 60 degrees-per-second, but not different from the fast velocity group in the trained limb. The (F) condition was significantly different in strength gains from the control at 450 degrees-per-second, but not different from the slow velocity group in the the trained limb. The conditions were not significantly different from each other in the trained limb at the test velocity of 210 degrees-per-second. The three conditions significantly different from each at the test velocity of 60 degrees-per-second in the trained limb. The conditions did not differ in strength at velocities of 210 and 450 degrees-per-second in the trained limb. The conditions did not differ in the cross-sectional area of the upper arm in the trained limb. The (S) and (F) training conditions improved (PT) by 12.36% and 18.84% at their respective training velocities of 60 and 450 degrees-per-second. These improvements were significantly (p<.05) larger than (C). The (S) and (F) training conditions also increased (PT) by 11.56% and 11.24% at the non-training velocity of 210 degrees-per-second (p<.05). Significant 10.77% (p<.05) improvement in (AG) was recorded in the (S) condition. No changes in (PT) were recorded in the contralateral limb within the three conditions. These data support the concept of limited (S) and (F) hi-directional (PT) overflow and (S) velocity hypertrophy enhancement. The presence of cross education (CE) was not supported by this investigation. / Master of Science

Despite the growing popularity of goal orientation research, three questions remain largely unanswered: (1) are there 3 factors of goal orientation or only 2; (2) what predicts goal orientation; and (3) is goal orientation domain specific? To help answer these questions, 177 undergraduates were given a questionnaire assessing, in both the academic and athletic domains, (a) learning, prove, and avoid goals, (b) self-perceived ability, ability, and implicit theories, and (c) high-school grade point average, intrinsic motivation, internal motivation, self-efficacy, locus of control, need for achievement, desire to win, and fear of negative evaluation. The results suggest that learning, prove, and avoid goals can be empirically distinguished, that they are domain specific, but that they are not predicted well by ability, self-perceived ability, or implicit theories. Discussion centers on the need for a pattern approach to the prediction of goal orientation and stresses the importance of examining the interactions among learning, prove, and avoid goals. The overriding conclusion, however, is that goal orientation is not a useful construct. / Master of Science

Yes / This study shows how highly branched poly(N-isopropyl acrylamide) (HB-PNIPAM) with a chain pendant solvatochromic dye (Nile red) could provide a fluorescence signal, as end groups bind to bacteria and chain segments become desolvated, indicating the presence of bacteria. Vancomycin was attached to chain ends of HB-PNIPAM or as pendant groups on linear polymers each containing Nile red. Location of the dye was varied between placement in the core of the branched polymer coil or the outer domains. Both calorimetric and fluorescence data showed that branched polymers responded to binding of both the peptide target (D-Ala-D-Aa) and bacteria in a different manner than analogous linear polymers; binding and response was more extensive in the branched variant. The fluorescence data showed that only segments located in the outer domains of branched polymers responded to binding of Gram-positive bacteria with little response when linear analogous polymer or branched polymer with the dye in the inner core was exposed to Staphylococcus aureus. / Innovate UK/Smith and Nephew Ltd. (UK) (TSB 103988) and by MRC (MR/N501888/2).

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type. / QC 20100929