Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a substrate. Comparisons with other ACPs included the following items: biological activity, pI, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule. ACPs from E. coli and Euglena have been shown to interact with melittin, a cationic peptide from bee venom. E. coli ACP is a small (Mr, 8847), acidic, Ca$\sp{++}$-binding protein which possesses some characteristics resembling those of regulatory Ca$\sp{++}$-binding proteins including interaction with melittin. Melittin inhibited activity of the nonaggregated FAS from Euglena using either E. coli or Euglena ACP as a substrate. The peptide also inhibited activity of the aggregated FAS from Euglena. Antibodies against melittin were raised. Anti-melittin inhibited activity of both the nonaggregated and aggregated FAS enzyme systems from Euglena relative to nonimmune antibody. Investigation of inhibition of the nonaggregated FAS enzyme system demonstrated that acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and keto-acyl-ACP synthetase activities were inhibited to different degrees by anti-melittin antibodies, while keto-acyl-ACP reductase and enoyl-ACP reductase enzyme activities were not inhibited.

To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol when cells were treated with TPA, an activator of PKC. To confirm that alkylglycerol was exerting an effect directly on PKC, alkylglycerol was shown to inhibit PKC activity in vitro in a dose dependent manner. Since PKC exists as a family of closely related isozymes, we have determined the effects of growth arrest and alkylglycerol treatment on PKC $\rm\alpha,\ \epsilon,\ and\ \zeta$ (expressed in MDCK cells). The active forms of PKC $\alpha$ and $\epsilon$ are lost early in the growth of MDCK cells during the endogenous accumulation of alkylglycerol and synthetic alkylglycerol inhibits the membrane form of PKC $\alpha$ and $\epsilon.$ However, alkylglycerol inhibits the TPA induced translocation of PKC $\alpha$ but not $\epsilon$ suggesting a differential inhibition among these isoforms. Neither TPA or alkylglycerol had any effects on the distribution of PKC $\zeta.$ To examine the effect of alkylglycerol on cell proliferation, Swiss 3T3 cells were used. GLC analysis shows that 3T3 cells accumulate alkylglycerol in a similar manner as MDCK cells. Since this accumulation occurs just prior to cell growth arrest, the effects of alkylglycerol on preconfluent cells was observed. Preconfluent cultures of 3T3 cells were treated with alkylglycerol on day 1 of growth. After 8 days of culture, the treated group showed a slower growth rate and saturation density. Furthermore, after these cells were reseeded in the absence of alkylglycerol, the original growth rate and saturation density returned. Thus alkylglycerol induces a decrease in cell proliferation without causing any detrimental effects. Similarly, alkylglycerol was found to inhibit the induction of mitogenesis by TPA (a PKC dependent pathway) and these effects were shown not to be stereospecific. To further investigate the effect of alkylglycerol on cell proliferation, the content of the monoglycerides in ras-transformed cells was analyzed. These cells have lost contact dependent growth arrest indicating a disruption of cell growth regulation. We observed a massive increase in the content of alkylglycerol during the culture of ras transformed cells. This increase is 3 fold higher than MDCK or 3T3 cells. This raises the possibility that alkylglycerol may be the end result of an increased number of cell-cell contacts. We have observed an increase in the accumulation of alkylglycerol in normal and ras-transformed cells. This accumulation is accompanied by a decrease in PKC activity and alkylglycerol was shown to be a potent in vitro inhibitor of PKC. Similarly, alkylglycerol was shown to inhibit PKC $\alpha$ under stimulation by TPA. Alkylgylcerol is a inhibitor of the TPA induced induction of mitogenesis and slows the growth rate of proliferating cultures of 3T3 cells. These results indicate that the endogenous ether-linked glycerolipid, alkylglycerol, is a regulator of cell proliferation through its inhibitory effects on protein kinase C. (Abstract shortened by UMI.)

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs element is intrinsically dynamic and may influence the stability of the surrounding genome. Thus, the stability of the L2Hs element was tested in a bacterial model system. A cloned 0.6 kb L2Hs element forms non-B-form structures in recombinant plasmids pN6 and pN2, which differ only in insert orientation. Instability of pN6 and pN2 plasmids was observed in serial propagation studies in which E. coli cells containing the plasmids were cultured every 24 hours for 28 days. The vector plasmid pTZ19U, as control, was found to be stable in all passages while the two L2Hs recombinants developed deletions of the L2Hs insert as well as adjacent vector sequences. The isolated deletion mutants have been characterized via restriction cleavage studies and sequencing to map the boundaries of the deletions. Direct repeats and potential stem-loop structures have been discovered at or within close proximity to the deletion boundaries. The data demonstrate that the L2Hs recombinants' unusual sequence features with potential for non-B-form secondary structures, influence genome stability via their involvement in generating errors during DNA replication and DNA repair.

There is solid evidence that complex human diseases can be caused by rare variants. Next generation sequencing technology has revolutionized the study of complex human diseases, and made possible detecting associations with rare variants. Traditional statistical methods can be inefficient for analyzing sequence data and underpowered. In addition, due to high cost of sequencing, it is also necessary to explore novel cost effective studies in order to maximize power and reduce sequencing cost. In this thesis, three important problems for analyzing sequence data and detecting associations with rare variants are presented. In the first chapter, we presented a new method for detecting rare variants/binary trait associations in the presence of gene interactions. In the second chapter, we explored cost effective study designs for replicating sequence based association studies, combining both sequencing and customized genotyping. In the third chapter, we present a method for analyzing multiple phenotypes in selected samples, such that phenotypes that are commonly measured in different studies can be jointly analyzed to improve power. The methods and study designs presented are important for dissecting complex trait etiologies using sequence data.

Thirteen N* states have been well-established according to the Particle Data Group, but some relativized quark models predict that many more N* resonances exist. Diquark models predict that the N* spectrum is limited by a correlated quark-pair in the nucleon, but there is strong evidence for the existence of the [Special characters omitted.] (1900)** resonance, which is absent in diquark models. Measuring the spectrum of N* states will provide valuable information on the relevant degrees of freedom within the nucleons. Most of the experimental searches for the N* states have been conducted in the πN channel. Some models of baryon decays predict that most of the unobserved N* states couple somewhat weakly to the πN channel, and that some couple non-negligibly to the KY, K*Y , and KY* channels. Measurements of the cross sections and polarization observables of strangeness photoproduction reactions can provide additional information on the spectrum of N* states. These measurements can be used in coupled-channel partial-wave analyses that can provide simultaneous constraints on the N* resonance parameters from several channels. These analyses can also take into account hadronic rescattering, which is predicted to have a large effect on the measured cross sections. However, to determine the isospin decomposition of the photo-transition amplitudes to these channels, photoproduction measurements are necessary on both the proton and the neutron. Measurements of the differential cross sections of the γn [arrow right] K* (892) 0 Λ and γn [arrow right] K + Σ*(1385) - reactions have been performed using data from the Jefferson Lab Hall B CLAS g13 experiment. No experimental cross section data have yet been published on the γn [arrow right] K* (892) 0 Λ reaction, and the only published cross section data on the γn [arrow right] K + Σ*(1385) - reaction are limited to forward angles, where t -channel K + and K* + exchanges are predicted to dominate. These cross sections are compared against theoretical models to study the channel interactions that give rise to their distributions. These reactions also have the same final state particles ( K + π - pπ - ), so studies of their potential interference were performed as well. A measurement of the γn [arrow right] pπ - cross section was also performed, and the agreement with published results within the uncertainties validated the integrity of the data and procedures used in this analysis.

Arsenic and uranium in the environment are hazardous to human health and require better methods for detection and remediation. Nanocrystalline iron oxides offer a number of advantages as sorbents for water purification and environmental remediation. First, highly uniform and crystalline iron oxide nanocrystals (nMAG) were prepared using thermal decomposition of iron salts in organic solutions; for the applications of interest in this thesis, a central challenge was the adaptation of these conventional synthetic methods to the needs of low infrastructure and economically disadvantaged settings. We show here that it is possible to form highly uniform and magnetically responsive nanomaterials using starting reagents and equipment that are readily available and economical. The products of this approach, termed the 'Kitchen Synthesis', are of comparable quality and effectiveness to laboratory materials. The narrow size distributions of the iron oxides produced in the laboratory synthesis made it possible to study the size-dependence of the magnetic separation efficiency of nanocrystals; generally as the diameter of particles increased they could be removed under lower applied magnetic fields. In this work we take advantage of this size-dependence to use magnetic separation as a tool to separate broadly distributed populations of magnetic materials. Such work makes it possible to use these materials in multiplexed separation and sensing schemes. With the synthesis and magnetic separation studies of these materials completed, it was possible to optimize their applications in water purification and environmental remediation. These materials removed both uranium and arsenic from contaminated samples, and had remarkably high sorption capacities -- up to 12 wt% for arsenic and 30 wt% for uranium. The contaminated nMAG is removed from the drinking water by either retention in a sand column, filter, or by magnetic separation. The uranium adsorption process was also utilized for the enhanced detection of uranium in environmental matrices. By relying on α-particle detection in well-formed and dense nMAG films, it was possible to improve soil detection of uranium by more than ten-thousand-fold. Central for this work was a detailed understanding of the chemistry at the iron oxide interface, and the role of the organic coatings in mediating the sorption process.

In this work, we prove three types of results with the strategy that, together, the author believes these should imply the local version of Hilbert's Fifth problem. In a separate development, we construct a nontrivial topology for rings of map germs on Euclidean spaces. First, we develop a framework for the theory of (local) nonstandard Lie groups and within that framework prove a nonstandard result that implies that a family of local Lie groups that converge in a pointwise sense must then differentiability converge, up to coordinate change, to an analytic local Lie group, see corollary 6.3.1. The second result essentially says that a pair of mappings that almost satisfy the properties defining a local Lie group must have a local Lie group nearby, see proposition 7.2.1. Pairing the above two results, we get the principal standard consequence of the above work which can be roughly described as follows. If we have pointwise equicontinuous family of mapping pairs (potential local Euclidean topological group structures), pointwise approximating a (possibly differentiably unbounded) family of differentiable (sufficiently approximate) almost groups, then the original family has, after appropriate coordinate change, a local Lie group as a limit point. (See corollary 7.2.1 for the exact statement.) The third set of results give nonstandard renditions of equicontinuity criteria for families of differentiable functions, see theorem 9.1.1. These results are critical in the proofs of the principal results of this paper as well as the standard interpretations of the main results here. Following this material, we have a long chapter constructing a Hausdorff topology on the ring of real valued map germs on Euclidean space. This topology has good properties with respect to convergence and composition. See the detailed introduction to this chapter for the motivation and description of this topology.

Dark matter, a non-luminous, non-baryonic matter, is thought to constitute 23 % of the matter-energy components in the universe today. Except for its gravitational effects, the existence of dark matter has never been confirmed by any other means and its nature remains unknown. If a hypothetical Weakly Interacting Massive Particle (WIMP) were in thermal equilibrium in the early universe, it could have a relic abundance close to that of dark matter today, which provides a promising particle candidate of dark matter. Minimal Super-Symmetric extensions to the standard model predicts a stable particle with mass in the range 10 GeV/c 2 to 1000 GeV/c 2 , and spin-independent cross-section with ordinary matter nucleon σ x ∠ 1 × 10 -43 cm 2 . The XENON100 experiment deploys a Dual Phase Liquid Xenon Time Projection Chamber (LXeTPC) of 62 kg liquid xenon as its sensitive volume, to detect scintillation ( S1 ) and ionization ( S2 ) signals from WIMP dark matter particles directly scattering off xenon nuclei. The detector is located underground at Laboratori Nazionali del Gran Sasso (LNGS) in central Italy. 1.4 km of rock (3.7 km water equivalent) reduces the cosmic muon background by a factor of 10 6 . The event-by-event 3D positioning capability of TPC allows volume fiducialization. With the self-shielding power of liquid xenon, as well as a 99 kg liquid xenon active veto, the electromagnetic radiation background is greatly suppressed. By utilizing the difference of ( S2/S1 ) between electronic recoil and nuclear recoil, the expected WIMP signature, a small nuclear recoil energy deposition, could be discriminated from electronic recoil background with high efficiency. XENON100 achieved the lowest background rate (∠ 2.2 × 10 -2 events/kg/day/keV) in the dark matter search region (∠ 40 keV) among all direct dark matter detectors. With 11.2 days of data, XENON100 already sets the world's best spin-independent WIMP-nucleon cross-section limit of 2.7 × 10 -44 cm 2 at WIMP mass 50 GeV/c 2 . With 100.9 days of data, XENON100 excludes WIMP-nucleon cross-section above 7.0 × 10 -45 cm 2 for a WIMP mass of 50 GeV/c 2 at 90% confidence level.

The biogenesis and maintenance of the entire endomembrane system is dependent upon membrane fusion proteins. Mounting evidence indicates that the integral membrane GTPase Atlastin is a membrane fusion protein involved in the homotypic fusion of the endoplasmic reticulum (ER) membrane suggesting a role in the biogenesis and maintenance of ER structure. I helped show that recombinant Drosophila atlastin is able to promote the fusion of synthetic membranes in vitro and that this fusion is dependent upon atlastin GTPase activity. The structure-function experiments presented here assist in elucidating domains required in the mechanism of atlastin mediated membrane fusion. ER homotypic fusion is dependent upon the self-association of Atlastin subunits in adjacent membranes to bring the bilayers into close molecular contact. Atlastin dimerization occurs in the presence of GTPγS but not GDP and stable dimerization is dependent upon a juxtamembrane middle domain three-helix bundle (3HB). The atlastin GTPase domain and 3HB form a potent soluble domain inhibitor of atlastin homotypic fusion, while the GTPase domain alone shows little inhibition. Designed GTPase domain mutations show that GTP binding and atlastin dimerization is insufficient to support fusion without GTP hydrolysis. Additionally, domain analysis of atlastin reveals that the C-terminal cytoplasmic domain of atlastin is absolutely required for membrane fusion, possibly through a protein-lipid interaction of an amphipathic alpha-helix. Genetic lesions in the human Atlastin-1 gene, SPG3A, result in a form of autosomal dominant hereditary spastic paraplegia (HSP). A better understanding of Atlastin function should lend significant insight into normal ER biogenesis and maintenance, as well as the pathology of human disease.

Photophysical, electronic, and compositional properties of single-walled carbon nanotubes (SWCNTs) and bulk nanotube samples were investigated together with graphene oxide photoluminescence. First, we studied the effect of external electric fields on SWCNT photoluminescence. Fields of up to 10 7 V/m caused dramatic, reversible decreases in emission intensity. Quenching efficiency was proportional to the projection of the field on the SWCNT axis, and showed inverse correlation with optical band gap. The magnitude of the effect was experimentally related to exciton binding energy, as consistent with a proposed field-induced exciton dissociation model. Further, the electronic composition of various SWCNT samples was studied. A new method was developed to measure the fraction of semiconducting nanotubes in as- grown or processed samples. SWCNT number densities were compared in images from near-IR photoluminescence (semiconducting species) and AFM (all species) to compute the semiconducting fraction. The results provide important information about SWCNT sample compositions that can guide controlled growth methods and help calibrate bulk characterization techniques. The nature of absorption backgrounds in SWCNT samples was also studied. A number of extrinsic perturbations such as extensive ultrasonication, sidewall functionalization, amorphous carbon impurities, and SWCNT aggregation were applied and their background contributions quantified. Spectral congestion backgrounds from overlapping absorption bands were assessed with spectral modeling. Unlike semiconducting nanotubes, metallic SWCNTs gave broad intrinsic absorption backgrounds. The shape of the metallic background component and its absorptivity coefficient were determined. These results can be used to minimize and evaluate SWCNT absorption backgrounds. Length dependence of SWCNT optical properties was investigated. Samples were dispersed by ultrasonication or shear processing, and then length-fractionated by gel electrophoresis or controlled ultrasonication shortening. Fractions from both methods showed no significant absorbance variations with SWCNT length. The photoluminescence intensity increased linearly with length, and the relative quantum yield gradually increased, approaching a limiting value. Finally, a strong pH dependence of graphene oxide photoluminescence was observed. Sharp and structured excitation/emission features resembling the spectra of molecular fluorophores were obtained in basic conditions. Based on the observed pH-dependence and quantum calculations, these spectral features were assigned to quasi-molecular fluorophores formed by the electronic coupling of oxygen-containing addends with nearby graphene carbon atoms.