The ability to bypass most cellular barriers to gain access to intracellular compartments has great potential in cell biology. The possibilities range from efficient delivery of macromolecules such as plasmids to small proteins and oligonucleotides that are sensitive to degradation. In biomedicine, easy access means enhanced cellular imaging and delivery of many therapeutics currently hampered by poor stability and cellular uptake. Carbon Nanotubes (CNTs) are attractive in these applications due to their efficient cellular uptake. While mode of entry of CNTs into cells is debatable, possibly their natural shape allows for their selective penetration across biological barriers in a non-destructive way, making them versatile as membrane permeating particles. The present study explores the diverse functionalities of CNTs including: 1) Efficient delivery of DNA into HeLa cells using vertically aligned MWNT arrays, 2) The use of Single Walled Carbon Nanotubes (SWNTs) as nano detoxifiers and 3) the design of SWNTs for efficient cellular uptake. Generally, vertically aligned nanoneedles have been used to influence the behavior and differentiation of various cell types. In the first work described in chapter 2, periodic high-density array MWNT nanoneedles is shown to support cell growth and penetrate into HeLa cells, making it ideal for use in cellular imaging and the efficient delivery of plasmid DNA into cells. Most importantly, we show that transfection with the MWNT substrate exhibited more uniformity in comparison to the commercially available lipofection procedure. Lipofection involves the formation of a complex of DNA and cationic lipids that interact with the cell via electrostatic interactions, leading to internalization, DNA escape into the cytosol, and the eventual transport into the nucleus. Functionalized CNTS have demonstrated great biocompatibility and potential for drug delivery in vitro. In the work described in chapter 3, we synthesized acid-oxidized and non-covalently PEGlyated SWNTs, which were reported previously for drug delivery purposes, and explored their potential for detoxification in the bloodstream. We investigated the binding of SWNTs to a pore-forming toxin pyolysin. The SWNTs were found to prevent toxin-induced pore formation in the cell membrane of human red blood cells. Quantitative hemolysis assay and scanning electron microscopy were used to evaluate the inhibition of hemolytic activity of pyolysin. Unlike HeLa cells, human red blood cells did not internalize oxidized SWNTs according to Raman spectroscopy data. Molecular modeling and circular dichroism measurements were used to predict the 3D structure of pyolysin (domain 4) and its interaction with SWNTs. The Tryptophan-rich hydrophobic motif in the membrane-binding domain of pyolysin, a common construct in a large family of cholesterol-dependent cytolysins (CDCs), showed high affinity for SWNTs. In the final two chapters, chapters 4 and 5, we focused on shorter CNTs (<70 nm) that have less length variations. This enabled the determination of several length related characteristics such as cellular uptake and distribution of SWNTs within between cells. Here, cellular uptake of two water-soluble SWNTs, Short SWNTs (S_SWNTs) and Ultra-Short SWNTs (US_SWNTs), was evaluated against various mammalian cells. Cellular entry of S_SWNTs (chapter 4), similar in dimensions to those reported in the literature, is shown to be affected by their hydrophilic corona and exhibit time-dependent nuclear accumulation. In contrast, US_SWNTs show no dependence of cellular entry on their hydrophilic exterior (chapter 5). Furthermore, intracellular localization and excretion of the US_SWNTs is observed to be cell type-dependent. Results presented in this work show the potential of CNTs as nano detoxifiers. We also use CNTs as vertically aligned nanoneedles and as colloids to efficiently traverse the plasma membrane. While CNTs as nanoneedles show the potential as an efficient means of transfecting mammalian cells, the use of S_SWNTs and US_SWNTs highlight some key observations including the physical and chemical properties (size, surface functionality) and cell type influence on cellular uptake and intracellular trafficking. These findings contribute to the interpretation of SWNT-cell interactions by providing a correlation between CNT length and cellular uptake and also cell type on trafficking of internalized SWNTs. With the realization of the enhanced permeability and retention effects, tumor vascular leakiness resulting from increased angiogenesis and vasoactive factors enhancing permeability at the diseased site, nanoparticles that have long circulation time have higher chance of accumulating at the diseased sites.

The goal of this study is to examine teacher feedback, learner uptake, and feedback perceptions in an adult Chinese as a Foreign Language (CFL) context. A 200-level Chinese reading course was observed for data collection. Participants included 13 students and one teacher. Thirteen class sessions (10 hours) were video-taped. A short survey, given at the end of each of the last six class sessions, was designed to elicit the teacher’s and the students’ perceptions of feedback frequency. The participants were not informed of the focus of this research at the beginning of data collection. Video-recorded data was fully transcribed and coded using Panova and Lyster’s (2002) feedback categorization. The teacher’s response to the survey was compared to that of the students’ regarding perceptions of feedback frequency. The results showed that the teacher provided feedback to 68.1% of all students’ errors. On average there was one feedback move every 2.4 minutes. All feedback types in Panova and Lyster’s model were present, and there were a few new moves, namely “asking a direct question,” “directing question to other students,” and “using L1-English.” A total of 245 teacher feedback moves occurred during the observation. Recasts accounted for 56.7% of all feedback moves, followed by metalinguistic feedback which accounted for 10.6%. Elicitation moves achieved the highest uptake rate (94.1%). Next, explicit correction and metalinguistic feedback had 88.9% and 53.8% uptake rate respectively. Concerning perceptions of feedback, the teacher was more accurate in perceiving four types of feedback while the students were accurate about three. It was concluded that recast was the predominant type of feedback in this study. Other explicit types of feedback were more successful in leading to learner uptake. The teacher and the students were generally not accurate in perceiving the frequency of each feedback type, due to the challenge of remembering the feedback move after the lesson had finished. The pedagogical implication is that teachers should consider a wide range of feedback techniques, especially more explicit types, to better engage students in a reading class. / Graduate

Riparian buffer zones, adjacent to waterways, may protect water quality by intercepting and removing nitrogen in runoff from agricultural land. This research comprised four parts: (1) a field study of nitrogen buffering by differently vegetated riparian zones in a United Kingdom (UK) sheep-grazed pastoral catchment; (2) a field study of surface and subsurface runoff hydrology, and nitrogen flux, in a UK riparian wetland; (3) a laboratory study ((^15)N tracer-isotope dilution) of microbial inorganic nitrogen production and removal processes in the UK riparian wetland soil; and (4) a laboratory microcosm study ((^15)tracer) of nitrate removal processes in bare and plant-inhabited (Glyceria declinata) New Zealand (NZ) riparian wetland soil. Dissolved organic nitrogen and ammonium were generally more important components of subsurface runoff than nitrate in the three UK riparian zones. All riparian zones were poor buffers having minimal effect on the nitrogen concentration of subsurface runoff. In the UK riparian wetland site subsurface (saturated zone) and surface 'preferential flow paths' typically conveyed large quantities of catchment runoff rapidly into, and across the site, and hindered nitrogen buffering. However, under low flow conditions, runoff-riparian soil contact increased and the wetland decreased the catchment nitrogen flux by 27%. In the UK riparian wetland soil most nitrate removal was attributable to denitrification (87- 100%) as opposed to dissimilatory nitrate reduction to ammonium (DNRA) (0-13%) and immobilisation (0-10%). Total ((^14)N+(^15)N) transformation rates for these processes were 1.3-47, 0.5-1.5 and 0.6-2.5 μg N g soil(^-1) hr(^-1) respectively. In the NZ riparian wetland soil Glyceria declinata assimilated 11-15% of nitrate but, more importantly, increased soil oxidation and altered the proportions of nitrate removal attributable to denitrification (from 29% to 61-63%) and DNRA (from 49 to <1%), but not immobilisation (22-26%). Denitrification and, thus, nitrogen buffering might be enhanced, in some riparian zones by increasing the extent of moderately anoxic soil with plants that release oxygen from their roots or with water table management.

The Vo sector of the vacuolar H+-ATPase is a multi-subunit complex that forms a proteolipid pore. The largest subunit in this complex is the a subunit which has four isoforms (a1-a4). The isoform(s) critical for secretory vesicle acidification has yet to be identified. Using a cell line derived from rat pheochromocytoma in which Voa1 and/or Voa2 had been down-regulated this study revealed that Voa1, and to a lesser extent, Voa2 are critical for acidifying dense-core vesicles (DCVs). The acidification defects resulting from down-regulation of Voa1 and Voa1/ Voa2 were suppressed by the expression of knockdown-resistant Voa1. Defects in DCV acidification resulted in reductions in their transmitter uptake and storage. Lastly, Ca2+-dependent peptide secretion appeared normal in Voa1 and Voa1/ Voa2 knockdown cells. . This study demonstrated that Voa1 and Voa2 cooperatively regulate dense-core vesicle acidification as well as transmitter uptake/storage, while they may not be critical for dense-core vesicle exocytosis.

The Vo sector of the vacuolar H+-ATPase is a multi-subunit complex that forms a proteolipid pore. The largest subunit in this complex is the a subunit which has four isoforms (a1-a4). The isoform(s) critical for secretory vesicle acidification has yet to be identified. Using a cell line derived from rat pheochromocytoma in which Voa1 and/or Voa2 had been down-regulated this study revealed that Voa1, and to a lesser extent, Voa2 are critical for acidifying dense-core vesicles (DCVs). The acidification defects resulting from down-regulation of Voa1 and Voa1/ Voa2 were suppressed by the expression of knockdown-resistant Voa1. Defects in DCV acidification resulted in reductions in their transmitter uptake and storage. Lastly, Ca2+-dependent peptide secretion appeared normal in Voa1 and Voa1/ Voa2 knockdown cells. . This study demonstrated that Voa1 and Voa2 cooperatively regulate dense-core vesicle acidification as well as transmitter uptake/storage, while they may not be critical for dense-core vesicle exocytosis.

Attention worldwide has been placed towards reducing the global carbon footprint. To this end the scientific community has been involved in improving many of the available methods of carbon capture and storage (CCS). CCS involves scrubbing flue gases of greenhouse gases and safely storing them deep underground. MOFs, a family of functionally tunable three dimensional nanoporous frameworks, have been shown to adsorb gases with great selectivity and capacity. Investigating these frameworks using computational simulations, although faster than in-lab synthetic methods, involves a tedious and meticulous input preparation process which is subject to human error. This thesis presents Dave's Occupancy Automation Package (DOAP),a software which provides a means to automatically determine the gas uptake of many three dimensional frameworks. By providing atomic coordinates for a unit simulation cell, the software acts to performs the necessary calculations to construct and execute a Grand Canonical Monte Carlo simulation, determining the gas uptake in a metal organic framework. Additionally an analysis of different convergence assessment tests for describing the end point of the GCMC simulation is presented.

Iron is a trace element required for a range of metabolic reactions in virtually all living organisms. Studies of prokaryotes, plants, yeast, and vertebrates have established detailed information on iron uptake and the role iron plays in metabolic processes. Iron is an essential growth requirement of schistosomes in vitro and schistosomes also express the highly conserved iron storage protein ferritin. However, studies into how this iron is taken up by the parasite have been neglected. This study aims to identify molecules involved in iron uptake and homeostasis in the human parasite Schistosoma japonicum. I have characterised two isoforms of a divalent metal transporter (DMT), a membrane bound protein of schistosomes. These DMTs have significant homology to the mammalian DMT1, the primary ferrous iron uptake protein of the intestinal brush border. Both schistosome isoforms displayed functional iron uptake by rescuing growth in a yeast strain deficient in iron uptake (fet3fet4). Interestingly schistosome DMT1 was localised to the tegument and not the gastrodermis of adult parasites, suggesting surface mediated iron uptake across the tegument. In physiological conditions, iron is abundant as largely insoluble ferric iron and hence ferric reductases are an essential component of iron uptake, reducing iron to the soluble ferrous form. Cytochromes b561 (Cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. Recent observations that Cyts-b561 may be involved in iron metabolism have opened new perspectives for their physiological function. Here, I have identified a new member of the cytochrome b561 family in Schistosoma japonicum that localises to the tegument of this trematode. Expression of the SjCytb561 in a Saccharomyces cerevisiae mutant that lacks plasma membrane ferrireductase activity (fre1fre2) was able to rescue the growth defect in iron deficient conditions, suggesting involvement in iron metabolism. Plasma membrane ferrireductase activities were also quantified using intact transformed yeast cells. These data further support the hypothesised tegumental uptake of host iron. Further, I have identified a putative schistosome transferrin. In mammals, transferrin is a glycoprotein responsible for binding and transporting iron in the bloodstream and delivering iron into cells via a specific transferrin receptor. Preliminary characterisation of the schistosome transferrin sequence has revealed it does not contain all the conserved amino acid residues associated with iron binding, with conservation seen only in the C-terminal lobe, not in both the N and C-lobes observed in mammalian transferrins. This difference makes it unclear whether the schistosome transferrin shares functional homology with its mammalian counterpart. In addition, no transferrin receptor has been identified to support an iron trafficking and uptake function, nor would this function be expected in an acoelomate organism. Further characterisation and localisation of this protein is required to elucidate its biological significance and function. The tegumental location of both the SjDMT1 and the SjCytb561 for the uptake of host iron make it possible to consider these proteins as potential vaccine candidates. A preliminary vaccination study with these proteins elicited only low to moderate protection from infection, and further studies are required to fully assess their potential. The data presented in this thesis provide evidence for surface-mediated uptake of iron by adult schistosomes, and represent the first characterisation of iron uptake proteins in any helminths. These studies show a novel method of iron uptake in schistosomes, and contribute to our understanding of how these parasites are able to survive and thrive by scavenging nutrients, in this case iron, from the host organism.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 112-130).

Thesis (Ph. D.)--Illinois State University, 2002. / Title from title page screen, viewed February 3, 2006. Dissertation Committee: Maarten E.A. Reith (chair), Hou Tak Cheung, Paul A. Garris, Stephen M. Lasley, Brian J. Wilkinson. Includes bibliographical references and abstract. Also available in print.

Thesis (M.S.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains vi, 69 p. Includes abstract. Includes bibliographical references (p. 30-39).