Dissertations / Theses on the topic 'Lipid Probes'

LDs are considered to be organelles with extremely low water content and high viscosity. Related to diseases such as diabetes, diabetes, cancer, etc., when the disease is abnormal, lipid droplets in cells will appear, so we have developed four types of lipid droplets. We designed and constructed a simple coumarin-absorbed p-nitrophenbutylethyl compound as a potential new organic biocatalyst for imaging groups. The internal projection belt shifts to the solar wavelength region. In addition, it is produced by the framework of the donor structure of the Fox also bridge. Stokes camera (100 nm, more than good LD, low biological toxicity and low biological toxicity and introduction. In addition, the biological probe Cou-LDs can also mark the emission of LDs in live zebras. We synthesized two new probes, LDP-1 and LDP-2, which showed a resolution of 4758 cm-1 and 3986 cm-1, respectively. In addition, the biological probes LDP1 and LDP show low biological toxicity and good specificity. These two probes are also suitable for life cycle monitoring of cell LD release in HeLa. At the same time, we have developed a new type of luminescent chemical sensor that can effectively mark the inside of the cell. In addition, the anti-interference, pH stability, and low biological toxicity of decoys have been deeply rooted in cell imaging and zebra fish imaging.
Ліпідні краплі (LD) вважаються органелами з надзвичайно низьким вмістом води та високою в’язкістю. Пов’язані з такими захворюваннями, як цукровий діабет, рак, тобто, коли хвороба є аномальною, у клітинах з’являться ліпідні краплі, тому ми розробили чотири типи ліпідних крапель. Розроблено просту п-нітрофенбутилетилову сполуку, що поглинає кумарин, як потенційний новий органічний біокаталізатор для груп візуалізації. Внутрішній проекційний спектр зміщується в видимій області світла. Крім того, сполуку виготовляють на основі донорського матеріалу. Камера Стокса (100 нм, більш ніж хороший LD, низька біологічна токсичність і низька біологічна токсичність і введення). Синтезовано два нових зонди, LDP-1 і LDP-2, які показали роздільну здатність 4758 см-1 і 3986 см-1 відповідно. Крім того, біологічні зонди LDP-1 і LDP-2 демонструють низьку біологічну токсичність і хорошу специфічність. Ці два зонди також підходять для моніторингу життєвого циклу вивільнення клітинної LD в HeLa. Розроблено новий тип люмінесцентного хімічного датчика, який може ефективно позначати внутрішню частину клітини.

The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length. Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores. A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles. The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly.
Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách. Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů. Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency). Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně.
This thesis has been elaborated within the framework of the Agreement on JointSupervision (co-tutelle) of an International Doctoral Degree Programmebetween Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.

This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.

Le ciblage, l'imagerie et le sondage spécifiques des membranes plasmiques et des organites intracellulaires peuvent être faits par des sondes fluorescentes à façon sensibles à la polarité. Ici, un nouveau fragment ciblant la membrane plasmique à été développé et testé dans cinq colorants cyanines, montrant d'excellentes performances en microscopie cellulaire et in vivo. Le fragment à été greffé à un fluorophore solvatochrome Prodan, donnant une sonde de membrane plasmique avec une sensibilité élevée à l'ordre lipidique. Le rouge de Nil, greffé aux fragments avec les chaînes alkyles C12 et C4, à donné deux sondes solvatochromes à membrane plasmique : NR12A pour la microscopie conventionnelle, et NR4A pour la microscopie à super-résolution PAINT. Le rouge de Nil avec des groupes ciblant les organites à donné un éventail de sondes sensibles à la polarité et à l'ordre lipidique dans les membranes des organites. Les sondes synthétisées trouveront des applications en bioimagerie, biologie cellulaire, biophysique ou mécanobiologie
Specific targeting, imaging and probing of cell plasma membranes and intracellular organelles can be addressed by rationally designed polarity-sensitive fluorescent probes. Here, a new efficient plasma membrane-targeting moiety was developed and tested in five cyanine dyes, showing excellent performance in cellular and in vivo microscopy. Next, the targeting moiety was grafted to a solvatochromic dye Prodan, yielding a plasma membrane probe with high lipid order sensitivity. Modifying a Nile Red using the moieties with varied alkyl chain lengths resulted in two solvatochromic plasma membrane probes: NR12A with high affinity to membranes for conventional microscopy, and NR4A, a low-affinity probe for PAINT super-resolution microscopy. Tethering Nile Red with organelle-targeted groups yielded an array of probes, able to sense polarity and lipid order in organelle membranes. The synthesized probes will find applications in bioimaging, cell biology, biophysics or mechanobiology

Conçues à partir d’une approche rationnelle, nous avons créé de nouvelles sondes membranaires permettant l’imagerie de l’organisation de la membrane plasmique cellulaire. Dans ce travail, nous avons d’abord développé un groupe d’outils, à partir du fluorophore solvatochrome Nile Red et de Black Hole Quencher-2, capable de marquer spécifiquement les domaines ordonnés et désordonnés (radeaux) en les identifiant par leur couleur d’émission. Les études cellulaires, à l’aide de ces sondes, suggèrent que la membrane plasmique est composée de deux phases distinctes. Puis dans le but de créer de nouvelles sondes basées sur Nile Red compatibles avec le sérum et fixables par formaldéhyde/glutaraldéhyde, nous avons modifié la sonde, préalablement développée, NR12S avec un groupement PEG ou amino, respectivement. Etonnamment, la sonde PEGylée est rapidement internalisée dans la cellule et le dérivé animo agrège avec l’agent fixant. D’un autre côté,basée sur Nile Red, nous avons conçu une sonde capable de détecter un récepteur donné et de visualiser son environnement lipidique. Initialement, nous avons obtenu des sondes capables d’allumer leur fluorescence en se liant sur le RCPG à l’ocytocine. Puis, nous avons conjugué NR12Spar l’intermédiaire d’un espaceur PEG(12) au ligand de l’intégrine, RGD. Les résultats préliminaires montrent que la molécule peut se lier à la membrane et détecter l’ordre lipidique, cependant les études cellulaires nécessitent un achèvement. Nous avons aussi travaillé sur des sondes membranaires fluorogéniques (turn-on) pour de l’imagerie multi-couleurs. Basées sur le fluorophore3-méthoxychromone, nous avons obtenu des sondes plus brillantes et plus photostables que la sonde développée originellement à partir de 3-hydroxychromone (F2N12S). Grâce à l’important déplacement de Stokes, elles permettent une imagerie de la membrane cellulaire avec une autofluorescence minimale dans la région spectrale bleue, compatible avec les marqueurs communs verts et rouges. Pour finir, basées sur le fluorophore squaraine, nous avons développé trois nouvelles sondes opérant dans la région rouge lointain, qui est particulièrement intéressante pour l’imagerie in vitro et in vivo. Ces sondes montrent une orientation parallèle avec la membrane lipidique, alors que les expériences cellulaires indiquent que seule la sonde avec deux ancres lipidiques est capable de marquer de façon stable la membrane plasmique. Ces sondes développées ici sont prévues pour être utilisées dans la recherche des radeaux lipidiques aussi bien que pour l’imagerie super-résolution et multi-couleurs de cellules vivantes
Based on rational molecular design, we design new membrane probes that enable fluorescence imaging of cell plasma membrane organization. In this work, we first synthesized a toolkit, based on solvatochromic Nile Red dye and Black Hole Quencher-2, that can stain specifically ordered and disordered lipid domains (rafts) and identify them by the emission color. Cellular studies with these probes suggested that the plasma membrane is composed of two distinct phases. Then,with the idea to make Nile Red-based probes compatible with serum medium and fixable by formaldehyde/glutaraldehyde, we modified previously developed probe NR12S with PEG and aminogroups, respectively. Surprisingly, the PEGylated probe is quickly internalized inside the cell and the amino-derivative aggregates with the fixing agent. On the other hand, based on Nile Red we designed probes capable to detect a given receptor and visualize its lipid environment. Initially, we obtained probes that can turn-on fluorescence on binding to the oxytocin GPCR receptor. Then, we conjugated NR12S through a PEG(12) spacer to the ligand of intergrin, RGD. The first data show that the molecule can bind to the membrane and detect the lipid order, though cellular studies have to be completed. We also worked on fluorogenic (turn-on) membrane probes for multi-color imaging. Based on blue 3-methoxychromone dyes, we obtained probes that are brighter and more photostable than the originally developed probe based on 3-hydroxychromone (F2N12S). Due to large Stocks shift, they enabled cell membrane imaging with minimal auto-fluorescence in the blue spectral region, compatible with common green and red probes. At the end, based on squaraine fluorophore, we developed three new probes operating in the far red region, which is also very interesting for in vitro and in vivo imaging. These dyes show a parallel orientation with the lipid membrane, while the cellular experiments point out that only the probe with two anchor groups is able to stain stably the plasma membrane. The probes developed here are expected to be used for lipid rafts research as well as for super-resolution and multi-color imaging of living cells

This thesis is focused on investigation of phospholipid-hyaluronan system. First, appropriate method for preparation of bulk solution of phospholipid/lipid and suitable fluorescence probe were chosen. Sonification was selected as a method for preparation of bulk solution and pyrene was chosen as a fluorescence probe. From the group of phospholipids lecithin was selected. Next to phospholipid, lipid with no phosphate group (DPTAP) was utilized for comparison, alternatively a mixture of lipid (DPTAP) and phospholipid (DPPC). Instead of hyaluronan another polyelectrolytes (sodium polystyrene sulfonate, sodium alginate) were used too. Measurements were performed in water environment and in phosphate buffer saline (PBS). All investigation was accomplished by fluorescence spectroscopy and dynamic light scattering.

Thesis advisor: Eranthie Weerapana
This thesis includes two projects: “Bacteria-selective borono-peptides” and “A split ligand for lanthanide binding: facile evaluation of dimerizing proteins”. In both projects, de novo designed molecules were synthesized, optimized and incorporated into peptides. These synthetic molecular tools allow selective targeting of bacterial cell membranes and analyzing the dynamic associations of membrane-embedded proteins. 1. Bacteria-selective borono-peptides As the antibiotic resistance continues to grow, bacterial infection becomes one of the major threats to global public health. Currently, almost all the bacteria targeting strategies employ non-covalent driving forces, including charge-charge interactions, hydrophobic interactions and the formation of hydrogen bonds, to achieve bacterial selectivity. Towards novel bacteria targeting molecules, we have recruited reversible covalent chemistry in the development of bacteria-selective peptides. Targeting the diol-rich environment of a bacterial surface, we have designed and synthesized several unnatural amino acids that contain boronic acid moieties. Taking advantage of the boronic acid-diol reaction and multivalency effect, our borono-peptides are found to selectively recognize bacteria over mammalian cells. The sensitivity of the binding event to carbohydrate competitors gives a safe and facile approach to regulate molecular association with bacterial cells. This design may find applications in the fields of bacterial detection, imaging and antimicrobial drug delivery. 2. A split ligand for lanthanide binding: facile evaluation of dimerizing proteins Protein dimerization is a ubiquitous phenomenon in biology and plays a critical role in transcription regulations and various signaling processes. Methods that allow facile detection and quantification of protein dimers are highly desirable for evaluating protein dimerization in physiology and disease. Meanwhile, luminescence of lanthanides is attractive for biological applications due to its long lifetime and sharp emission profiles. We have developed a split lanthanide binding ligand that allows facile evaluation of dimerizing proteins. The fast lanthanide–ligand (dis)association allows us to monitor the dynamic behavior of dimerizing proteins. We have demonstrated the successful application of our assay on both soluble and transmembrane proteins in complex biological milieu. The split lanthanide ligand is cysteine reactive, and therefore should be readily applicable to a variety of proteins of interest
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Thesis advisor: Jianmin Gao
Lipid reporters are key signaling molecules in a number of biological processes ranging from apoptosis in mammalian cells to novel resistance mechanisms in pathogenic bacteria. Developing probes to target these lipids is a worthy endeavor, especially when better reporters could mean lives saved. This is particularly true considering new antibiotic resistant pathogens emerge every year with evolving lipid compositions. To combat these pathogens and prevent a potential global pandemic, it is imperative to continue the development of novel and innovative probes/drugs to meet this daunting challenge. To fulfill this demand, we must continue to establish new strategies, enhance current technologies and advance scientific understanding. Only by pushing the boundaries of what is currently possible will we remain one step ahead of these diseases. Diseases like mcr-1 positive bacteria, first documented in 2016, remain largely uncontested. Herein, we seek to expand the available probes specific to key lipid reporters for phosphatidylserine, lysyl-phosphatidylglycerol, and phosphoethanolamine lipid A. Cyclic phage libraries were first utilized to target phosphatidylserine, ultimately producing weak binders. Refining our phage display libraries to include reversible covalent warheads allowed for the identification of more potent lipid reporters. In doing so, we have created the tools necessary to interrogate the unique resistance mechanisms expressed by these drug-resistant pathogens. A strong correlation was observed between peptides binding mcr-1 positive strains, LPS modification on the surface of these bacteria, and level of colistin resistance. To our knowledge, these peptides are the only probes capable of demonstrating this correlation. We surmise that the methods discussed here will pave the way for better diagnostic tools for these resistant pathogens. A recurring method of resistance among gram-positive and gram-negative bacteria has been to decorate their surface with positive amines to repel cationic antimicrobial peptides. As such, our current APBA library and the libraries in development in the Gao lab would be ideally suited to target these and other undiscovered resistance mechanisms
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

The cellular membrane is a complex biological entity, far from being an inert assembly of protein and lipids which separates cells from the surrounding environment. A multitude of biological processes, ranging from active transport of ions into and out of the cell, to the immune response, are regulated at the level of the plasma membrane. The understanding of their molecular basis is among the central goals of modern biological research. In order to dissect the complexity of actual cell membranes, which involves a very complex network of intermolecular interactions, a “divide and conquer” strategy proves very useful. To this end, researchers try to isolate molecules from complex biological contexts to understand their function in simple model systems under controlled conditions. A variety of model membranes have thus been developed in order to gain insight into membrane processes. This approach has resulted in a deeper knowledge on how lipids and proteins interact and how these interactions govern the function of cellular membranes. In the recent past in fact, a connection has been established between the lateral structure of the plasma membrane and its biological function. Furthermore, a large range of biophysical techniques have been used to characterize protein-lipid microdomains. For example, atomic force microscopy (AFM) is a powerful technique which allows a highly detailed topographical characterization of lipid domains in physiological conditions. While AFM imaging offers an extremely high spatial resolution, up to the nanometer scale, the limited image acquisition speed (minutes) can pose a severe drawback in adequately studying fast dynamic processes. On the other hand, fluorescence based imaging techniques are much faster (10-3-100 s), but certainly lack the high spatial resolution that AFM offers. FCS in particular can also provide information about dynamic processes, like diffusion of fluorescent membrane components. For these reasons, implementing a combination of the above mentioned techniques on the same sample (e.g. cell membrane models) would prove extremely beneficial in the complete dynamic and structural characterization of molecular interactions. . The work described in this thesis can be summarized in two main points: i) the development of a novel combined approach of atomic force microscopy (AFM), laser scanning imaging (LSM), and fluorescence correlation spectroscopy (FCS) and ii) the study of the effects of ceramide in the lateral organization of model plasma membranes. We described one of the first simultaneous applications of AFM and FCS on biologically relevant systems. More specifically, model membranes showing complex phase separation were investigated with a combined approach of AFM, confocal fluorescence imaging, force measurements and FCS, based on commercially available instruments. AFM conveys information about the structural and mechanical properties of the different lipid phases. Different membrane domains can be distinguished based on height difference, elastic properties and line tension as measured by the AFM tip. Simultaneous optical measurements offer the correlation of these data in real time with the partition behavior and diffusion of fluorescent lipids and proteins. We established a clear link between the local membrane viscosity, probed by FCS, and the lipid-lipid interactions involved in line tension, probed by AFM force measurements. An example of a significant drawback circumvented by the AFM-FCS approach involves the use of AFM micromanipulation to eliminate unwanted interactions between lipid particles — similar to intra-cellular vesicles found in vivo experiments — and the membrane, which usually result in distorted FCS autocorrelation curves. Finally, the combined application of AFM and FCS on membrane-anchored proteins reconstituted in lipid bilayers has been instrumental in clarifying inconsistencies that arose in work that focused solely on either AFM or fluorescence techniques. We have shown that, in the case of proteins diffusing in the plane of the membrane, AFM can unambiguously detect only a small immobile fraction. Furthermore, since AFM detection of proteins might be facilitated by high local membrane viscosity (e.g. in ordered lipid phases), the measurement of protein partition between disordered and ordered membrane domains might be biased toward the latter. In this case, the use of FCS as a complementary technique allows a more thorough investigation and deeper understanding of the system of interest. The second part of this thesis dealt with the study of complex lipid mixtures which are used to model the putative lipid/proteins domains in cells, called “rafts”. Firstly, we proved how the combined fluorescence imaging/AFM approach is useful in general for studying supported lipid membranes and the role of lipid domains in biological contexts. We investigated the effect of environmental stress on biological membranes and the protective effects of several substances. Our experimental approach was shown to be a new valuable method to visualize the dehydration damage and its effects on the lateral organization of lipid domains. Our results demonstrated that disaccharides like trehalose or sucrose are effective in protecting lipid membranes, not only on a macroscopic scale — preserving the overall integrity of the bilayer — but also on a microscopic scale, preventing the clustering of microdomains. These phenomena are interesting in the context of biological damage to living cells which need to be stored for long time, like organs to be transplanted or blood platelets. Finally, a large section of this thesis focused on the effects of a specific lipid called “ceramide” on the lateral organization of proteins and lipids in the plasma membrane. Ceramide is produced by cells in several situations, like bacterial infections or apoptosis. As consequence of ceramide production in vivo, the local concentration and the dynamic behavior of lipids and membrane receptors are supposed to exhibit strong variations. In order to understand the molecular mechanisms responsible for these effects, we applied a combination of AFM, FCS and fluorescence imaging on simple model membranes containing ceramide. We could show for the first time that, in presence of raft-like Lo/Ld phase separation, physiological quantities of ceramide induced the formation of a highly ordered gel phase, constituted of ceramide and sphingomyelin. The enzymatic production of ceramide was monitored both in supported and in free-standing bilayers. In the second case, ceramide production was connected to selective vesicle budding from the raft-like phase. Since short-chain analogues are often used in both medical applications and biochemical research to mimic the effect of long-chain ceramides, we investigated the effect of chain-length on ceramide-induced membrane reorganization. We could show that only long-chain ceramides (C18 and C16) form highly ordered domains. Interestingly, FCS measurements indicated that the physical properties of the Lo raft-like domains are hardly affected by the presence of ceramide domains. Furthermore, the increased thickness of the Ld phase — as measured by AFM — and its higher viscosity — as measured by FCS — strongly support the hypothesis of ceramide-induced cholesterol displacement from rafts. On the other hand, short-chain ceramides showed completely different biophysical properties that lead to a destabilization of the raft domains, possibly acting as surfactants between the different lipid phases. Our findings contribute to the explanation of in vivo experiments where short-chain ceramides inhibit cell signaling by disrupting the lipid order in the plasma membrane. We have so far demonstrated that ceramide plays a fundamental role in lipid-lipid interactions. In a physiological context, it is also known to produce dramatic effects in living cells. Since a majority of the processes in vivo are thought to be governed by the activity of proteins, it is highly likely that ceramide not only affects lipid organization but also modifies protein-protein and protein-lipid interactions to produce its effects. To test this hypothesis, we reconstituted several membrane proteins in lipid bilayers containing Ld, Lo, and ceramide-rich domains. We were able to show that some membrane proteins are sorted into ceramide-rich domains. More specifically, the raft-associated proteins we tested were enriched in the highly ordered ceramide-rich domains, while the Ld-associated components were excluded from them. Furthermore, the inclusion of any membrane component in ceramide-rich domains is directly connected to a dramatic reduction of its in-plane diffusion. In an in vivo context, such a reorganization of membrane receptors might be used by the cell to alter the signaling process, for example, by i) separating raft receptors from inhibitors with lower raft affinity, ii) bringing both raft-associated receptors and raft-associated signaling molecules into contact, or iii) stabilizing the interactions between a receptor and its ligand by decreasing their diffusion coefficients. In conclusion, this thesis describes a novel combination of AFM, LSM, and FCS for the investigation of the lateral organization of biological membranes. Our results show that this approach applied on model membranes of increasing complexity is an effective tool for understanding the molecular mechanisms behind the organization of biological membranes. This report opens up new possibilities for further investigation in living cell membranes using the same methodology we have described.

As a hydrophilic biopolymer, a DNA molecule is surrounded by water molecules in aqueous solution. The charge hopping mechanism indicates the competition between radical cation quenching by water molecules and migration along DNA partially determines the distance and efficiency of charge transport in DNA. Lipid can effectively bind DNA to induce hydrophobic environment around the DNA helix and reduce the water contact with bases in the DNA duplex. Therefore, the effect of water molecules on charge transport can be studied by comparison between nature DNA and DNA-lipid complexes. We synthesized several cationic lipids with various lengths of dialkyl chain (2, 8, 18) and spermine (Sp4+) binding core in this research, which posses strong DNA binding affinity due to their multi-charged spermine head-groups. Among those, C8GlySp4+ and C2GlySp4+ can form stable complex with DNA oligomer in aqueous solution, characterized by time dependent UV and CD spectrometry. C2GlySp4+ showed the similar inhibition on oxidative damage in GG steps as spermine while C8GlySp4+ demonstrated much more significant prohibitive effect at the same concentration. Since all the lipids bear the same binding core, they should afford the similar binding affinity towards DNA duplexes. we attributed the observation to the longer length of dialkyl group in C8GlySp4+, which can more effectively shield the DNA duplex from the water molecules than either spermine or C2GlySp4+. A kinetic model based on phonon-assist polaron hopping mechanism was proposed to rationalize the experimental results. The finding may give insight on the protection of DNA oxidative damage by reducing the access of the water molecule to DNA duplex and may have potential impact on the application of DNA as conducting biopolymer and protection of DNA in biological system.

Biological research has benefitted greatly from the advent of omic methods. For many biomolecules, mass spectrometry (MS) methods are most widely employed due to the sensitivity which allows low quantities of sample and the speed which allows analysis of complex samples. Improvements in instrument and sample preparation techniques create opportunities for large scale experimentation. The complexity and volume of data produced by modern MS-omic instrumentation challenges biological interpretation, while the complexity of the instrumentation, sample noise, and complexity of data analysis present difficulties in maintaining and ensuring data quality, validity, and relevance. We present a corpus of tools which improves quality assurance capabilities of instruments, provides comparison abilities for evaluating data analysis tool performance, distills ideas pertinent in MS analysis into a consistent nomenclature, enhances all lipid analysis by automatic structural classification, implements a rigorous and chemically derived lipid fragmentation prediction tool, introduces custom structural analysis approaches and validation techniques, simplifies protein analysis form SDS-PAGE sample excisions, and implements a robust peak detection algorithm. These contributions provide improved identification of biomolecules, improved quantitation, and improve data quality and algorithm clarity to the MS-omic field.

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by which pathogenic keratin aggregations in EI and epidermal protein deficiencies in ARCI patients may be mitigated by retinoids. Furthermore, the vivid crosstalk between proteins incriminated in ARCI suggests that these enzymes operate along a common metabolic pathway essential for producing barrier lipids in stratum corneum. Any abrogation of this production may cause barrier failure, hence resulting in a compensatory hyperkeratosis characteristic of congenital ichthyosis.

De nombreux processus biologiques liés aux membranes cellulaires lipidiques sont encore très mal connus. La présence d'eau et d'ions à l'interface influence les propriétés structurelles et dynamiques de la bicouche lipidique. Les techniques de fluorescence sont très utiles pour étudier les membranes en raison de la grande sensibilité des sondes à leur environnement. Nous avons utilisé la technique de relaxation de solvant (SR) pour explorer l'hydratation et la mobilité de l'eau. Nous avons également réalisé des calculs quantiques (QM) et des dynamiques moléculaires (DM) pour étayer nos expériences. Les résultats SR montrent qu'un petit cation (Na+) est très attiré par la membrane et augmente sa rigidité à l'opposé des cations (NH4+, Cs+) plus gros. Les anions (CI04-, SCN-) s'adsorbent à l'interface plus facilement que Cl-. Ces anions changent la mobilité et l'hydratation des têtes polaires des lipides de la bicouche. Les études SR de la zone hydrophobe de la membrane montrent que les processus de relaxation sont ici très complexes. lis reflètent des processus rapides intramoléculaire (relaxation de torsion, transferts de charge) et des processus intermoléculaires lents. Les calculs QM ont permis de créer les champs de force de trois sondes fluorescentes (Prodan, Laurdan et C-laurdan). Les simulations DM ont permis de déterminer les positions des sondes dans une membrane DOPC. La modélisation reproduit correctement les résultats SR, en particulier les temps de relaxation : de l'ordre de la ps en solvant aqueux et de la ns dans la membrane. Les simulations MD sont complémentaires des méthodes SR et permettent de surveiller le comportement de molécules uniques
Many biologically important processes and phcnomena in lipid membranes are still not fully understood. The presence of ions and water molœules has a significant influence on the structural and dynamical properties of lipid bilayers. Fluorescent techniques are versatile tools for studying the lipid membranes, because the fluorescence emission is strongly sensitive to dye environment. We have conducted fluorescent solvent relaxation (SR) experiments to explore the hydration and mobility properties in lipid membranes in the presence of different chaotropic ions. We have also carried out Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations for supporting the SR experiments. SR experiments show that small cation (Na+) is attracted to the membrane and increases rigidity ofbilayer, while larger cations (NH/, Cs+) should not. Large anions (CI04·, SCN') adsorl, at the membrane interface more easily than smaller ones (Cl') and significantly change tl!e mobility and hydration of the headgroup region oflipid bilayer. SR study ofhydrophobic part of the membrane show that SR processes are complex there and reflect botl!: faster, intramolecular (torsional relaxation or fonnation of charge transfer state) and slower, intermolecular (SR) relaxation processes. QM calculatiom were used to create force-field for three fluorescent dyes (Prodan, Laurdan and C-laurdan). MD simulations allow detennining position of the dye in the lipid membrane in the ground state and after excitation and reproduce correctly SR timescale- ps in water and ns in the membrane. MD simulations extend the capabilities of SR method and allow observing the behaviour of individual molecules

Protein–lipid binding interactions play crucial roles in various physiological and pathological processes, making it very important to study these interactions at the molecular level. However, investigation of these interactions is complicated by several issues, including the inherent complexity of membranes as well as the diverse mechanisms by which proteins interact with the membrane surfaces. As a result, many of these interactions remain poorly characterized. Synthetic probes are useful tools employed for studying protein–lipid binding interactions. This thesis will detail the design and synthesis of metabolically stabilized analogues of various signaling lipids, which mimic the natural species and are not easily modified by enzymes present in biological systems. A modular approach is employed for synthesizing these lipid probes, giving access to a wide range of derivatized lipid probes that can then be used for several studies. Although a wide variety of metabolically stabilized lipid analogues have been synthesized, their activity has not yet been characterized and quantified in detail. So, there is a great need to synthesize biologically active phosphorothioate and phosphonate analogues of various signaling lipids in order to properly characterize and compare the binding affinities and activity of these analogues. Synthesis of metabolically stabilized lipid analogue would take us one step closer towards understanding protein–lipid interactions in biological systems and in trying to find answers to the myriad of questions pertaining to these systems.

Signaling lipids such as diacylglycerol (DAG) and the phosphatidylinositol polyphosphates (PIPns) play crucial roles in numerous cellular pathways. However, characterization of their activities is hindered by the complexity of associated signaling pathways and of the membrane environment. To address this issue, we have developed lipid probes that are effective for characterizing biological events using different applications, including activity-based probing (PIPns and DAG) and microarray analysis (PIPns). The activity-based probes have been applied to label receptor targets in multiple cancer cell proteomes through photocrosslinking followed by click reactions. The probes were found to label several proteins, as judged by on-gel fluorescence, and labeling was abrogated through various controls, such as heat denaturation and competition. Proteomic studies have been successfully performed to identify protein targets through biotin enrichment followed by mass spectrometric analysis. For microarray analysis, functionalized PIPn probes were synthesized and applied to develop a high throughput microarray analysis to measure protein-lipid binding affinity. These approaches will be invaluable for characterizing PIPn/DAG-regulated events and their involvement in disease. The design, synthesis and application of these lipid probes are included in this dissertation. In addition, the design and synthesis of other lipid probes are discussed, such as bis(monoacylglycero)phosphate (BMP), and lysophophatidylcholine (LPC) analogs.

Indiana University-Purdue University Indianapolis (IUPUI)
The cell membrane serves as a barrier between the interior and exterior of a living cell. Its main structural component is the lipid bilayer, which is composed of various kinds of lipids that segregate into domains. These lipid domains, distinguished in composition and physical properties from the bulk lipids that surround them, are believed to modulate the function of resident proteins by providing an appropriate lipid environment. Polyunsaturated fatty acids (PUFA) are a type of fatty acid that contain multiple C=C double bonds. They have a lot of health benefits, which may originate in part due to their incorporation into lipids in the plasma membrane. Hypotheses that PUFA-containing lipids laterally separate into domains and/or modulate the structure of existing domains have been raised to explain the fundamental role played by PUFA. In our research, we use molecular dynamics (MD) simulations to simulate model membranes composed of PUFA-containing phospholipids and to investigate their interaction with cholesterol and vitamin E that are influential membrane constituents. The presumptive function for vitamin E in membranes is to protect PUFA against oxidation. Although the chemistry of the process is well established, the role played by the molecular structure that we address with atomistic molecular dynamics (MD) simulations remains controversial. We compared the behavior of vitamin E in lipid bilayers composed of 1-stearoyl-2-docosahexaenoylphosphatidylcholine (SDPC, 18:0-22-6PC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) via all-atom MD simulations at 37° C. SDPC represents a PUFA-containing lipid, and SOPC serves as monounsaturated control. From the calculation of van der Waals energy of interaction between vitamin E and fatty acid (FA) chains, we found higher probability that the PUFA chains surround the chromanol head group on vitamin E. This is further demonstrated by probability density maps of acyl chains around vitamin E molecules. Also, an ability to more easily penetrate deep into the PUFA containing bilayer of vitamin E is detected by faster flip-flop rate of vitamin E observed in the SDPC bilayers. These results showed that the high disorder of polyunsaturated docosahexaenoic acid (DHA) chains allows vitamin E to easily tunnel down into the bilayer and often brings the PUFA chains up to the surface of the bilayer, improving the likelihood that the reactive (hydroxyl) group on vitamin E would encounter a lipid peroxyl radical and terminate the oxidation process. Thus, the simulations indicate that the molecular structure of vitamin E supports its role as an antioxidant in a PUFA-containing membrane. A subsequent study on the partitioning of vitamin E into PUFA-containing lipids was done by analyzing the binding energy of vitamin E in the corresponding lipid bilayer. The binding energy is obtained from the potential of mean force (PMF) profile of vitamin E alone the membrane normal direction (z), which is calculated from umbrella sampling MD simulations. We found the binding in SDPC is smaller in SOPC, indicating that vitamin E does not prefer PUFA-containing phospholipids. The flip-flop rate was also estimated from the PMF profile, confirming that vitamin E flip-flops across the SDPC bilayer more easily than the SOPC bilayer. From the simulations it was noted that the membrane deforms as vitamin E is pulled out, which suggests interactions between the phospholipids contribute to the binding energy of the vitamin E. In a final study, a comparison was made between the effect on membrane organization of the three types of long chain omega-3 (n-3) PUFA found in fish oils: eicosapentaenoic acid (EPA, 20:5), DHA (22:6) and docosapentaenoic acid (DPA, 22:5). MD simulations were run on lipid bilayers composed of 1-stearoyl-2-eicosapentaenoylphosphatidylcholine (EPA-PC, 18:0-20:5PC), 1-stearoyl-2-docosapentaenoylphosphatidylcholine (DPA-PC, 18:0-22:5PC), SDPC (DHA-PC, 18:0-22:6PC) and, as a monounsaturated control, SOPC (OA-PC, 18:0-18:1PC) in the absence and presence of cholesterol. By analyzing the physical properties such as membrane order and thickness, we found all three n-3 PUFAs disorder the membrane. The disordering is greatest with EPA and least with DPA. Unique among the n-3 PUFA-containing membranes, there is region of high order in the upper portion of the DPA chain. The PUFA-containing lipids were found to less favorably interact with cholesterol compared to the OA-containing lipid, which is caused by their disorder. We speculate that differences between DPA, DHA and EPA might potentially modulate their effect on lipid domain formation.

碩士
國立成功大學
材料科學及工程學系碩博士班
101
We study the indentation test of a free-standing lipid bilayer by molecular dynamics simulation, and discuss the effect of the interaction between the probe and the lipids on the bilayer structure. By calculating the local physical quantities of the lipid bilayers in the indentation process, we confirmed that the cause of bilayer rupture is not only because bilayer local thinning make the free energy of the lipid tails increase, bilayer local extension also make the lipid tails exposure to solvent and further increase the free energy of the lipid tails. These two reasons both increase the probability of micro voids formation of the lipid bilayers. In the retraction process, we found that if the interaction between the probe and the lipid tails is insufficient to make lipids adsorbed on the probe, the lipid molecules tend to rearrange to form hydrophilic bilayer edges in the rupture regions and eventually leave a hydrophilic pore in the bilayer, and no residual lipids were found on the probe. Conversely, if the lipid tails are adsorbed by the probe in the retraction process, the lipid bilayers can revert to a free-standing state, and the physical quantities of the lipid bilayers before and after the indentation are almost identical. Besides, we found that there are some residual lipids were adsorbed by the probe, and the number of the residual lipids depends on the interaction between the probe and the lipid head groups, the stronger the interaction is, the more number of residual lipids were adsorbed by the probe. These results can help us to find a suitable pharmacologically nanoinjectors. Furthermore, we investigated the relationship between the force-indentation curve and the interaction between the probe and the lipids. As expected, we observe that when the interaction between the probe and the lipid tail is stronger, the lager maximum attractive force can be measured in the retraction process at the same indentation speed. These results show that we can use the force –indentation curve obtained from AFM experiments to define the magnitude of the interaction between the probe and the lipids, and help us to select the proper probe materials in the relevant AFM experiments.

Lipidomics and metabolomics are powerful tools for the examination of cellular metabolism and physiology. Methods for lipid analysis need to be developed that begin with small samples and do not overly dilute or disperse the sample in the separation process. Microchips provide a platform for interfacing lysis of small cell populations with on-chip solid phase extraction for isolating lipid samples to generate high quality mass spectra from very small samples. Chapter 1 of this dissertation presents a novel method for small scale lipidomics of bacterial cells by microchip based extraction coupled with untargeted profiling of glycerophospholipids using nanoelectrospray ionization mass spectrometry. Chapter 2 and 3 focus on the development of LC-MS/MS methods to study biological pathways. In Chapter 2, I describe a method for analysis of the phospholipids metabolite, GroPIns, in the medium of the pathogenic yeast Candida albicans. This method was applied to aid in the characterization of the GroPIns transport protein, Git1, in C. albicans. Chapter 3 extends the studies of part two and describes an efficient method based on HILIC-MS/MS for the separation and quantification of five lipid-related extracellular metabolites in yeast Saccharomyces cerevisiae. This newly developed methodology was successfully applied to determine the extracellualr levels of glycerophosphoinositol, glycerophosphocholine, glycerol 3-phosphate, inositol and choline in wild type and mutant strains.
Bayer School of Natural and Environmental Sciences
Chemistry and Biochemistry
PhD
Dissertation

Carbohydrate–protein interactions play vital roles in various biochemical processes such as signal transduction and cell surface recognition events. The clustering of carbohydrates into dense domains such as lipid rafts regulates recognition by multivalent receptors (i.e. lectins). These regions are known to play important roles in biological processes such as cellular transduction and trafficking. In order to characterize the clustering of glycans on cell surfaces, detection of domains with high carbohydrate density is of great interest. In this thesis, we present the work based on a modular strategy to design and synthesize boronic acid-based carbohydrate receptors, which are termed as boronolectins because of their similarly in functions with lectins, in order to understand the molecular basis of carbohydrate–protein interactions. These receptors will then be employed for binding studies with carbohydrate based guests and catechol derivatized diol target molecules in order to study the binding interactions between the boronic acid receptors and diol moieties present in guest molecules. A second project that is described in this thesis is based on developing diacylglycerol-based lipid probes, which could be employed for studying protein-lipid binding interactions. Due to involvement of protein-lipid binding interactions in the onset of various pathophysiological conditions, it is of paramount importance to investigate these interactions at the molecular level. DAG (diacylglycerol) represents an important class of signaling lipids and members of the Protein kinase C (PKC) family are described as the main responsive receptors of DAG. PKCs are known to be involved in tumorigenesis. In order to elucidate the exact correlation between PKC activity and carcinogenesis, it will be beneficial to design and synthesize DAG-based lipid analogs. In this thesis, hence, a modular strategy to design and synthesize a class of DAG-based lipid analogs by appending reporter groups such as polyaromatic fluorophores in the sn-1 acyl chains via the traceless Staudinger ligation is described. In both the projects, the synthetic strategy adopted is based on a modular design in order to generate a common scaffold which undergoes modification at the last step to generate a class of fluorophore tagged analogs to be employed for studies.

Indiana University-Purdue University Indianapolis (IUPUI)
The details of Polarization Modulation Near-Field Scanning Optical Microscopy (PM-NSOM) are presented. How to properly calibrate and align the system is also introduced. A measurement of Muscovite crystal is used to display the capabilities of the setup. Measurements of supported Lβʹ 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers are presented, emphasizing how it was tooled in exploiting the anisotropic nature of the acyl chains. A discussion of how the effective retardance (ΔS = 2π( n_e-n_o )t/λ) and the direction of the projection of the acyl chains (θ) are measured simultaneously is given, (where t is the thickness of the bilayer and λ is the wavelength of light used). It is shown from ΔS the birefringence (ne-no) of the bilayer is determined, by assuming the acyl chain tilt with respect to the membrane's normal to be ϕ ≈ 32. Time varying experiments show lateral diffusions of ~ 2 x 10-12 cm2/s. Temperature controlled PM-NSOM is shown to be a viable way to determine the main phase transition temperature (Tm) for going from the gel Lβʹ to liquid disorder Lα state of supported DPPC bilayers. A change of ΔS ~ (3.8 +/- 0.3 mrad) at the main phase transition temperature Tm (≈41^o C) is observed. This agrees well with previous values of (ne-no) and translates to an assumed <ϕ> ~ 32^o when T < Tm and 0^o when T > Tm. Evidence of supper heating and supper cooling will be presented, along with a discussion of the fluctuations that occur around Tm. Finally it is shown how physical parameters such as the polarizability are extracted from the data. Values of the transverse (αt) and longitudinal (αl) polarizabilites of the acyl chains are shown to be, αt = 44.2 Å3 and αl = 94.4 Å3, which correspond well with the theoretical values of a single palmitic acid (C16) αt = 25.14 Å3 and αl = 45.8 Å3.

碩士
國立中興大學
食品科學系
93
Abstract Marketing Mei-Gin is a highly concentrated and dark black product made from squeeze juice of premature mei (Japanese apricot) via filtration. This product has been generally recognized as a food with several functional properties. In this study, Taiwan mei was used as raw materials to make Mei-Gin by traditional method which combined heating and concentration. Changes in quality during processing were observed and functional property for the final Mei-Gin was investigated by biological test of rat feeding. The results were as follows. 1. Heating and concentration were two necessary processes to obtain Mei-Gin of traditionally viscous and dark black appearance. The solution of diluted Mei-Gin which was concentrated to 10-fold in acidity by combined heating and concentration and then re-hydrated to original juice showed 46.95 for total color difference (ΔE) value. The value was quite high compared to that of circulated heating at normal pressure (3.62) and vacuum concentration (3.19). 2. Scavenging efficiency of DPPH for mei juice gradually increased with heating and concentration. The final Mei-Gin product was 33.24% higher than original juice, while no obvious changes for circulated heating at normal pressure method and little decrease for vacuum concentration method. The sediment fraction obtained from the diluted Mei-Gin solution by centrifugal fractionation showed 32~53% higher in DPPH scavenging efficiency original juice while decrease was observed for supernatant fraction. The changes became much more obvious with heating and concentration indicated that availably functional components were further produced during processing. 3. Diets mixed high cholesterol (0.2% cholesterol) with various concentrations (0.05%, 0.5%, 5%) of Mei-Gin were feed to rats. The blood biochemical assessment after 2, 4, and 8 weeks showed: No regular changes in both GPT and GOT;  Total cholesterol (TC) reduced from 210.92 to about 183 mg/dL at the 4th week, however, the decrease was not proportional to the concentrations Mei-Gin and was not significantly different with that of the 2nd and 8th week;  Total triglyceride (TG) obviously related to concentration of Bainiku-ekisu, it reduced from 115.03 to 70.75 mg/dL (closed to the normal group, 69.67 mg/dL) for the 2nd week, while from 119.32 to 47.2 mg/dL for the 4th week, and from 141.83 to 53.8 mg/dL for the 8th week;  No difference was observed in HDL for the 2nd week, but obviously increased for both 4th and 8th week though was not in proportion to the Mei-Gin concentrations;  There were no differences in LDL for any experimental groups at any weeks.

This thesis concerns the role of nutrients in cognitive and behaviour problems associated with child attention deficit hyperactivity disorder (ADHD). Study 1 investigated relationships between Conners' ADHD Index ratings, fatty acid deficiency symptoms (FADS), and cognitive performance in a normal population of children. Studies 2 and 3 comprised a 30 week intervention trial investigating effects of n-3 PUFA supplementation on ADHD symptoms in 7-12 year old children with high ADHD scores.