Niveoscincus ocellatus is an important species in historical analyses of the evolution of viviparity because it is the species upon which the type II chorioallantoic placenta was based. Here we describe the net nutrient uptake across the placenta of N. ocellatus for comparison with other species of skinks with complex placentae. N. ocellatus is highly placentotrophic, with neonates being 1.68-times larger in dry matter than the fresh eggs. There is an increase of nitrogen from 6.3 ± 0.2 mg to 9.2 ± 0.6 mg, and ash from 3.8 ± 0.3 mg to 6.7 ± 0.6 mg. The increase in ash is made up by a more than two-fold increase in the amounts of calcium, potassium and sodium. There is no significant difference in lipids in the neonates compared to fresh eggs, so considerable lipid must have crossed the placenta to provide energy for embryonic development. N. ocellatus is significantly more placentotrophic than Niveoscincus metallicus, which also has a complex chorioallantoic placenta. Discovery of substantial placentotrophy in this genus confirms that two lineages of Australian lygosomine skinks (represented by the genera Pseudemoia and Niveoscincus) have evolved this pattern of embryonic nutrition and supports the hypothesis that the evolution of reptilian placentotrophy involves specialisations in addition to structural modifications of the chorioallantoic placenta.

Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work. To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells. A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain. Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.

Retention of lipid-soluble components can be increased in a curd matrix by using emulsions rather than the direct addition of fortified oil. The retention of vitamin D_3 was increased in a model system with fortified emulsions (emulsifier: dairy proteins) 96-97% compared to 62-72% control. Retention of fortified emulsions (78%) remained greater than the control (58%) in small-batch Cheddar cheese. Bilayer emulsions were evaluated to increase retention of lipid-soluble components even further. Physicochemical characteristics of the bilayer emulsions were evaluated prior to curd inclusion. Nonfat dry milk (NDM) was used as the primary emulsifier at 1wt% dairy proteins. Polysaccharides (iota-carrageenan, low-methoxyl [LMp] and high-methoxyl pectin) and gelatin were secondary layers. Secondary emulsions formulation was 2.5 wt% oil, 0.5 wt% protein, and 0.2 wt% secondary biopolymer. Emulsions were adjusted to pH 3, 5, and 7 after homogenization. Factors that influence stability are biopolymer concentration, droplet size/distribution/charge (zeta-potential), and viscosity. iota-Carrageenan was the most stable, independent of pH, of all the emulsions. This increased stability was a consequence of the affinity of the protein layer and iota-carrageenan through the additional homogenization step. LMp was also stable at pH 7 due to calcium bridging, which correlates with the increased viscosity. The microstructure of the emulsions was examined using scanning electron microscopy. A strong correlation was found between emulsion instability and the presence of thick webbing, due to excess biopolymer, as seen in the micrographs. Stable emulsions were likely to have distinct droplets without a thick web. The exception was gelatin (pH 3), which still had individual droplets but was unstable due to depletion flocculation. The retention of lipid-soluble substances using secondary emulsion was evaluated in a model curd matrix between primary (50:50 fortified: non-fortified oil) and secondary (100% fortified oil) emulsions. There was no significant difference (α=0.05) in retention of “fortified” oil between primary and secondary emulsions; however, the same fortification level was obtained using secondary emulsions using half the oil. Curd made with 0.01M CaCl_2 had overall lower retention than curd with no additional calcium. Secondary emulsions could be used to fortify various gel matrices (e.g., curd, yogurt, and tofu). Marketing possibilities are endless after preliminary evaluation.

This dissertation represents an attempt to understand the mechanism of the action of pesticides derived from chlorinated phenols (pentachlorophenol, pentachlorobenzenethiol, and 2,4,5-trichlorophenol) in lipid membranes; specifically, the kinetics of pesticide induced hydrogen ion transfer in lecithin-cholesterol membranes and its relationship to uncoupling activity in energy transducing membranes. Information on pesticide induced charge transport in membranes was obtained from the measurements of steady state and transient membrane conductance and membrane potentials as a function of the composition of the aqueous phase surrounding the membrane and of membrane composition. In addition, a systematic theoretical study of a series of membrane transport models was performed in order to elucidate the various aspects of membrane transport kinetics and to make predictions relevant to the interpretation of experimental results. Based on the theoretical results, two kinetic schemes of membrane transport were proposed to explain the experimental results; one for pentachlorophenol and 2,4,5-trichlorophenol, and another for pentachlorobenzenthiol. The schemes differ in the proposed mechanism of charge transfer across membrane water interface. Some conclusions regarding the rate limiting step in the charge transport process are drawn. Measurements of membrane surface potentials based on the microelectrophoretic method reveal that the density of ionized pentachlorophenol at the membrane surface can be predicted from the Langmuir adsorption model, provided that electrostatic repulsion between pentachlorophenol ions adsorbed at the membrane and free ions in the aqueous phase is taken into account. The fact that the adsorption of positively charged tetraphenylarsonium ions and negatively charged salicylate ions modify the membrane charge transport induced by 2,4,5-trichlorophenol, can be explained by electrostatic arguments. The 2,4,5-trichlorophenol, can be explained by electrostatic arguments. The ability of pentachlorobenzenethiol and of chlorinated phenols to induce proton transport in membranes is regarded as the origin for both the membrane electrical conductivity and their toxic action in energy transducing membranes. We have found similarities between the pH dependence of pentachlorobenzenethiol induced membrane conductance and the pH dependence of the pentachlorobenzenethiol uncoupling activity found in mitochondria, as reported in the literature. We have also found that the level of pentachlorophenol concentration at which the membrane conductance increases above the background level corresponds to the onset of reduction of carbon uptake by algae.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: SMOF lipid™ infusion containing soybean oil, medium-chain triglycerides, olive oil, and fish oil has been approved and recommended for use in adults receiving parenteral nutrition (PN). Research shows that SMOF lipid infusion is safe for use in post-operative and critically ill patients. Improved patient outcomes are linked to SMOF lipid use over traditional soybean oil lipid emulsions. Objective: The purpose of this study was to determine the effect of SMOF lipid infusion on incidence of infection, ICU length of stay, hospital length of stay, and mortality in adult trauma patients as compared to parenteral nutrition utilizing 100% soybean oil emulsion or a lipid-free, dextrose and amino acid administration. Methods and Analysis: A retrospective chart review was conducted for adult trauma patients admitted to Eskenazi Health’s surgical intensive care unit (SICU) that received PN from May 2017 to May 2020. Data were collected from the electronic health record and trauma registry. Results: Twenty-nine patients were included who met study criteria: 17 patients in the traditional lipid cohort and 12 in the SMOF lipid cohort. The length of PN therapy was comparable between the traditional and SMOF ILE groups, 13.9 days (± 11.5) and 13.3 days (± 14.3) respectively. All 12 patients in the SMOF lipid cohort received intravenous lipid emulsion (ILE) compared to 42% (n=7) of traditional lipid group patients (p=0.001). SMOF treatment group were provided 100% of estimated energy needs via PN compared to an average of 94% (± 9.7) of estimated energy needs in the traditional lipid treatment group (p=0.036). Incidence of infection during initial hospitalization was significantly lower in the SMOF treatment group (n=3, 25%) compared to the traditional lipid treatment group (n=13, 76%). Mortality was decreased in the SMOF treatment group (0%) when contrasted to the Intralipid treatment group (23.5%), p=0.04. Conclusion: Patients receiving SMOF lipid emulsion within PN therapy had better clinical outcomes compared to those receiving Intralipid soybean-lipid emulsion or a dextrose and amino acid administration.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20287号 / 医科博第78号 / 新制||医科||5(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 岩井 一宏, 教授 萩原 正敏, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

TGFβ ligands, activin and myostatin have been shown to stimulate insulin production and secretion. Antagonists, Follistatin (FST) and Follistatin like 3 (FSTL3) were partially and fully ablated, respectively, creating hyperinsulinemic mice with fatty liver. Much research has surfaced on the connection between hepatic steatosis and hepatic insulin resistance. We present two different models, each with a different mechanism behind the development of fatty liver. FST288-only mice have increased synthesis of mRNA and proteins responsible for hepatic triglyceride (TG) uptake, while our double mutants have increased synthesis of mRNA and proteins responsible for TG synthesis. This alteration was likely independent of hepatic insulin resistance as livers from both mouse lines were insulin sensitive. Experiments conducted in this study to realize the causal factor of hepatic steatosis can be performed on adipose and muscle tissues in the future to better characterize the phenotype.

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The Amyloid Cascade hypothesis provides a molecular-level mechanism for the etiology of Alzheimer’s Disease (AD) and proposes a central role for the genesis and aggregation of Aβ protein. Aβ protein is the product of cleavage of the amyloid precursor protein (APP), a single pass transmembrane protein, by secretases and is found in a variety of isoforms, with longer isoforms being linked to the early onset of AD. The isoform distribution is dependent on membrane environment, mutations, and post-translational modifications. Lipid rafts are characterized by lipids induced into the liquid ordered phase by cholesterol, enhancing membrane thickness and lateral lipid density. Protein preference for rafts can control protein kinetics, and has been implicated in determining whether APP is processed by α– or β-secretase in the plasma membrane. In addition to inducing lipid rafts, cholesterol is hypothesized to directly modulate APP, the C-terminal fragment of APP (C99), and γ-secretase structure and function via direct interaction. To date, the molecular details involved in these fundamental events involved in Aβ genesis have yet to be resolved using experimental approaches, suggesting a critical role for computation. This thesis presents the results of investigations of lipid phase separation and cholesterol and their effects on C99 using molecular dynamics simulation. To gain insight into the nature of lipid rafts, studies characterizing the simulation system sizes required for observation of phase separation, exploring the effect of cholesterol concentration on phase separation and lipid phases, and examining the applicability of different lipid and cholesterol models for the simulation of lipid phases and protein structure were performed. To gain insight into the fundamental properties of C99, studies exploring the structure of full-length C99, the interaction of cholesterol with C99 in various mutational states, the effect of membrane thickness on the C99 extramembrane domains, and the structure of C99 monomer and dimer were performed. Taken together these studies advance our molecular-level understanding of the nature of cholesterol, the role of cholesterol in lipid phase separation, the effect of cholesterol on C99, and the structure of the full-sequence C99 monomer and dimer that play a critical role in the evolution of AD.