No description available.

Solid-state NMR (ssNMR) has matured into a versatile method to provide structural information, probe protein dynamics and detect small molecule binding and -protein interaction of a variety of biomolecular assemblies including amyloid fibrils, viral particles and membrane proteins. Membrane proteins embedded in liposomes are natural targets for ssNMR as their native states are solids. Magic angle spinning (MAS) ssNMR studies using moderate spinning frequencies provide detailed structural information and probe subtle conformational change. Development of fast magic angle spinning ssNMR enables proton-detection which increases sensitivity and facilitates protein dynamics measurements. In this dissertation, we applied moderate and fast MAS ssNMR to study potassium ion channel and protein dynamics Chapter 1 will introduce concepts and theory of solid-state NMR pulse sequences and experiments. Chapter 2 will discuss the application and perspectives of solid-state NMR to membrane protein systems. In Chapter 3, we test an allostery mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery. In Chapter 4, I optimize protein expression, purification and reconstitution into native environment protocols of a bacterial potassium transporter, KtrB. In chapter 5, methods and experimental details of setting up 60 and 40 kHz fast MAS ssNMR are discussed. With fast MAS ssNMR setup, multidimensional NMR experiments with higher sensitivity could be collected on a perdeuterated sample with less sample mass required. In Chapter 6, we employ fast MAS ssNMR to measure bulk and residue site-specific 15N and carbonyl 13C relaxation of microcrystalline ubiquitin. Carbonyl R1ρ relaxation profiles provide additional information on protein backbone dynamics.

Thesis: Ph. D., Joint Program in Oceanography (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / While aerobic respiration is typically invoked as the dominant mass-balance sink for organic matter in the upper ocean, many other biological and abiotic processes can degrade particulate and dissolved substrates on globally significant scales. The relative strengths of these other remineralization processes - including mechanical mechanisms such as dissolution and disaggregation of sinking particles, and abiotic processes such as photooxidation - remain poorly constrained. In this thesis, I examine the biogeochemical significance of various alternative pathways of organic matter remineralization using a combination of field experiments, modeling approaches, geochemical analyses, and a new, high-throughput lipidomics method for identification of lipid biomarkers. I first assess the relative importance of particle-attached microbial respiration compared to other processes that can degrade sinking marine particles. A hybrid methodological approach - comparison of substrate-specific respiration rates from across the North Atlantic basin with Monte Carlo-style sensitivity analyses of a simple mechanistic model - suggested sinking particle material was transferred to the water column by various biological and mechanical processes nearly 3.5 times as fast as it was directly respired, questioning the conventional assumption that direct respiration dominates remineralization. I next present and demonstrate a new lipidomics method and open-source software package for discovery and identification of molecular biomarkers for organic matter degradation in large, high-mass-accuracy HPLC-ESI-MS datasets. I use the software to unambiguously identify more than 1,100 unique lipids, oxidized lipids, and oxylipins in data from cultures of the marine diatom Phaeodactylum tricornutum that were subjected to oxidative stress. Finally, I present the results of photooxidation experiments conducted with liposomes - nonliving aggregations of lipids - in natural waters of the Southern Ocean. A broadband polychromatic apparent quantum yield (AQY) is applied to estimate rates of lipid photooxidation in surface waters of the West Antarctic Peninsula, which receive seasonally elevated doses of ultraviolet radiation as a consequence of anthropogenic ozone depletion in the stratosphere. The mean daily rate of lipid photooxidation (50 ± 11 pmol IP-DAG L⁻¹ d⁻¹, equivalent to 31 ± 7 [mu]g C m⁻³ d⁻¹) represented between 2 and 8 % of the total bacterial production observed in surface waters immediately following the retreat of the sea ice. / by James R. Collins. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21715号 / 薬科博第106号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 講師 樋口 ゆり子 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Development of liposome formulation of an amphiphilic anticancer peptide using the ANTS/DPX leakage assay. The effects of lipid composition on the liposomes' resistance to an amphiphilic cyclic peptide c[KS.S.S.KWL W] were studied by the ANTS/DPX leakage assay. One or more unsaturated acyl chains in the phospholipids, small phospholipid headgroup size, the presence of cholesterol, and the presence of PEG-lipid were demonstrated as critical parameters to stabilize the liposome membrane. A liposome formulation of the peptide comprising POPE/POPC/cholesterol/C16 mPEG 2000 ceramide (20.8:31.2:40:8, mol%) was thereby developed with a peptide-encapsulation efficiency of 47.8%. The liposomal cyclic peptide exhibited dose-dependent toxicity to MCF7 human breast cancer cells and stability under incubation. Design, construction and in vitro characterization of a hydrazone-based convertible liposomal system for anticancer drug delivery. A novel PEG-lipid, PEG2ooo-Hz-DHG, with an acid-labile hydrazone linker between the PEG2ooo head group and the lipidic DHG moiety was synthesized. PEG2000-Hz-DHG was relatively stable at normal physiological pH 7.4, but hydrolyzed more quickly at tumor interstitium pH 6.5-7.0 and endosomal/lysosomal pH 5.0. A novel pH-sensitive "Convertible Liposome System" (CLS) was constructed comprising PEG2ooo-Hz-DHG, positively charged lipid DOTAP, and the zwitterionic phospholipid POPC (8:15:77, mol%). CLS converted from neutrally charged "stealth" liposome to positively charged liposome at tumor interstitual pH owing to the hydrolysis ofPEG2ooo-Hz-DHG. The doxorubicin-encapsulated CLS that had been pre-incubated at pH 6.5 for 30 h exhibited more intensive binding and higher toxicity to Bl6-Fl0 murine melanoma and MDA-MB-435S human breast cancer cells than doxorubicin encapsulated in pH-insensitive stealth liposome.

Many attempts have been made to make cancer therapy more selective and less detrimental to the health of the patients. Nanoparticles have emerged as a way to solve some of the problems of traditional chemotherapy. Nanoparticles can provide protection for the therapeutic from degradation or clearance, as well as protection to healthy tissue from the damaging effects of chemotherapy drugs. Researchers are pursuing different strategies but all have the same goals of improving the outcomes of cancer patients. The field of controlled release of drugs has increased significantly in hopes of better treating diseases like cancer. Improved control of drug release has great potential for improving patient outcomes. Still there exist certain barriers such as circulation time, cell specificity, and endosomal escape.In this study a novel drug delivery vehicle was studied in vitro. The novel construct consisted of a liposome containing perfluorocarbon emulsions—an eLiposome—that was activated by ultrasound to break open on demand. Two targeting moieties were attached to the eLiposome to increase cell specificity and induce endocytosis. These studies determined the localization of eLiposomes in vitro using flow cytometry and confocal microscopy. Results indicated that eLiposomes modified with a targeting moiety attached to HeLa cells to a greater extent than non-targeting eLiposomes. Confocal images indicated localization of eLiposomes around the membrane of cells. Flow cytometer results indicated that ultrasound does in fact disrupt the eLiposomes but evidence of significant delivery to the cytoplasm was not obtained. However cells that were incubated with eLiposomes for 24 hours showed over 60% of the cells had green color association indicating eLiposome uptake.

Petroleum Technology Development Fund (PTDF) Nigeria / The full text will be available at the end of the embargo period: 21st March 2026

No description available.

No description available.

No description available.