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In binary systems, surfactant molecules can self-assemble into a large variety of structures depending on their chemical structure, concentration and temperature. The properties and stability of the phases, their coexistence regions and the formation of metastable structures is of great importance not only for fundamental understanding, but also for applications in many fields including industry and medicine. This thesis presents studies of the equilibrium and non-equilibrium behaviour of two widely used surfactant systems. The understanding of the equilibrium behaviour of an aqueous surfactant system is often incomplete or partly incorrect, which is caused by experimental difficulties, long equilibration times and the occurrence of long-lived metastable states. By applying a set of complementary techniques and recording changes on different length scales, the equilibrium phase diagram of the surfactant didodecyldimethylammonium bromide (DDAB) in water has been studied and amended. Differential scanning calorimetry has been used to obtain thermodynamic parameters. The structure of phases and biphasic regions have been characterised by small angle X-ray scattering and microscopy, while the conformational properties of the surfactant molecules have been investigated using Raman spectroscopy combined with computational methods. The effects of impurities have been studied using analytical techniques and a sufficient purity of the samples could be ensured. As a result of the studies, a new crystalline phase which exists at temperatures below 16°C was found. This phase replaces the frozen lamellar phase (Lβ) in the previously reported phase diagram. The Lβ phase has been found to be a long-lived metastable phase. The amended phase diagram has been tested by studying phase transitions along isoplethal and isothermal paths. All experimental results could be explained in terms of the new phase diagram. The study of phase transition along isoplethal paths focused on the transition between the new crystalline phase (XWn) coexisting with a dilute monomer solution (W) and the lamellar phase (Lα). This transition was (except for a single composition DDAB≈3% DDAB) a non-isothermal transition involving the phase sequence: XWn +W → XWn + Lα → Lα upon heating and Lα → overcooled Lα → XWn +W upon cooling. The structural changes within the phases and their relative ratios could be characterised using small angle X-ray scattering, microscopy and Raman spectroscopy. During the dissolution of lamellar phases along an isothermal path, multilamellar wormlike interface instabilities (so called myelins) were found to grow from the lamellar/water interface into the water. The growth of these myelins as well as changes in the lamellar phase have been investigated using optical microscopy and direct observation. This has provided detailed quantitative information on the dynamics of myelin growth and the effect of the initial structure of the lamellar phase on the myelin growth. The dependence of the growing rate on surfactant concentration could be explained in terms of a previously reported model in which the osmotic pressure was stated to be the driving force for the myelin kinetics. It has been found that for lamellar phases in coexistence with a sufficient amount of crystals, the myelin growth could be suppressed. Preliminary measurements of a tertiary system, where the pure lamellar phase of DDAB was mixed with a crystalline phase formed by dioctadecyldimethylammonium bromide (DODAB), a DDAB analogue, were carried out. The myelin growth has also been studied for a second system, the non-ionic surfactant triethylene glycol monododecyl ether (C12E3), known for its formation of myelins of great stability. The optical methods were extended to confocal microscopy, resulting in a 3D image of the myelin formation, providing detailed quantitative information on myelin growth as well as on myelin size.

Zusammenfassung Das Myelinprotein P0 stellt eine zentrale Komponente für die Stabilität und Funktionalität der Myelinscheiden des peripheren Nervensystems dar. Mutationen des P0-Proteins führen zu verschiedenen, schwer behindernden peripheren Neuropathien wie der Charcot-Marie-Tooth- oder der Dejerine-Sotas-Erkrankung. Wir haben das Tiermodell der P0-Knock-Out-Mäuse verwendet, um im Vergleich zu den C57BL/6-Wildtyp-Tieren Selektionsmechanismen des P0-spezifischen T-Zell-Repertoires zu untersuchen. Dazu wurde eine Reihe von überlappenden 20-mer-Peptiden benutzt, die die gesamte Aminosäuresequenz von P0 abdeckten. Mit Hilfe dieser Peptide wurde ein sog. „Epitop-Mapping“ der H2-Ab-restringierten T-Zell-Antwort durchgeführt. Auf diese Weise konnte das P0-Peptid 5 (Aminosäure 41-60) in der extrazellulären P0-Domäne als immunogene Determinante identifiziert werden. Dieses immunogene Peptid wurde dann für Untersuchungen der Toleranzmechanismen verwendet und zeigte, dass in P0-Knock-Out-Mäusen ein hochreaktives P0-spezifisches T-Zell-Repertoire vorliegt, während es in Wildtyp-Tieren inaktiviert ist und so Selbsttoleranz erzeugt wird. Die Toleranzerzeugung in Wildtyp- und heterozygoten P0 +/- Mäusen hängt nicht von der Gen-Dosis ab. P0 ist ein gewebespezifisches Antigen, dessen Expression normalerweise auf myelinisierende Schwann-Zellen beschränkt ist. Die klassischen Vorstellungen zu Toleranzmechanismen gegenüber gewebsspezifischen Antigenen schrieben diese vor allem peripheren Immunmechanismen zu. Durch den erstmaligen Nachweis von intrathymischer Expression gewebsspezifischer Antigene wie P0 konnten wir bestätigen, dass für P0 offensichtlich die Expression deutlich weiter verbreitet ist, insbesondere auch auf Thymus-Stroma-Zellen. Unter Verwendung von Knochenmarkschimären haben wir weitere Untersuchungen durchgeführt, wie Knochenmarks-abstammende Zellen im Vergleich zu nicht-hämatopoetischen Zellen Toleranz gegenüber P0 erzeugen können. Unsere Befunde zeigen, dass Knochenmarks-abhängige Zellen nicht ausreichen, um völlige Toleranz zu erzeugen. Zusätzlich wurde eine P0-Expression auf anderen Geweben wie dem Thymus benötigt, um komplette Toleranz zu erhalten. Wir identifizierten ein kryptisches P0-Peptid 8 und zwei subdominante P0-Peptide 1 und 3. Während das Peptid 8 sowohl in Wildtyp- als auch Knock-Out-Mäusen erkannt wurde, wurden die Peptide 1 und 3 in Wildtyp-Mäusen nicht als Immunogen erkannt. Die genannten Peptide wurden verwendet, um eine experimentelle autoimmune Neuritis (EAN) zu erzeugen. Mit keinem der experimentellen Ansätze konnten wir klinische Zeichen einer EAN generieren, allerdings mit dem Peptid 3 doch Entzündung im peripheren Nerven beobachten. Es werden zukünftig weitere Untersuchungen benötigt, um P0-spezifische T-Zell-Linien zu etablieren und so mit höherer Effizienz eine EAN zu erzeugen. Unsere Untersuchungen sprechen dafür, dass bei gentherapeutischen Ansätzen bei erblichen Neuropathien vorsichtig und schrittweise vorgegangen werden muss, da mit sekundärer Autoimmunität und damit Inflammation im peripheren Nerven zu rechnen ist. / Summary Myelin protein zero (P0) is a key myelin component in maintaining the integrity and functionality of the peripheral nervous system. Mutated variants are the cause for several disabilitating peripheral neuropathies such as Charcot-Marie-Tooth disease or Dejerine –Sotas syndrome. Using P0 knockout mice - a mouse model for these diseases - together with their wt counterparts on C57BL/6 background we studied the shaping of the T-cell repertoire specific for P0 in the presence and in the absence of this protein during the ontogeny of T-cells. Our approach was to use a series of overlapping 20-mer peptides covering the entire amino acid sequence of P0. This series of P0 peptides was employed for epitope mapping of the H2-Ab restricted T cell response. Thus, P0 peptide 5 (P0 41-60) in the extracellular domain of P0 was identified as the main immunogenic peptide. The immunogenic peptide containing the core immunodominant determinant in the P0 sequence was employed in studies of tolerance, revealing a highly reactive P0 specific T-cell repertoire in P0 ko mice while in wt mice the high avidity repertoire was inactivated in order to ensure self tolerance. In wild type and heterozygous P0 mice tolerance is not dependent on gene dosage. P0 is a tissue specific antigen whose expression is limited to myelinating Schwann cells. The classical view on tolerance to tissue specific antigens attributed this role to peripheral mechanisms. Driven by the finding that intrathymic expression of tissue-specific antigens is a common occurrence, we confirmed that “promiscuous” expression on thymic stroma holds true also for myelin P0. In addition, using bone marrow chimeras we investigated the capacity of bone marrow derived cells versus nonhematopoietic cells to induce tolerance towards P0. Our findings show that bone marrow derived cells although tolerogenic to some degree are not sufficient to mediate complete tolerance. P0 expression on cells with origin other than bone marrow showed to be sufficient and necessary to induce sound tolerance. We identified one cryptic (P0 peptide 8) and two subdominant epitopes (P0 petides 1, and 3). P0 peptide 8 was reactive in both wt and P0 ko mice. Peptides 1 and 3 were immunogenic in P0 ko but not in wt mice. Several P0 peptides including the immunogenic peptide 5 were involved in direct and adoptive transfer EAN studies. None of them induced clinical signs of EAN. Immunization with P0 peptide 3 did induce inflammation of the peripheral nerves reflected by the infiltration of macrophages and CD3 positive cells. More studies involving highly P0 specific T-cell lines are needed to characterize the P0 induced EAN. Our findings may have direct implications for secondary autoimmunity and inflammation in peripheral nerves developing after correcting the P0 genetic defect by gene therapy in aforementioned diseases.

Thesis advisor: Thomas N. Seyfried / GM1 gangliosidosis is a glycosphingolipid lysosomal storage disease caused by a genetic deficiency of acid b-galactosidase (β-gal), the enzyme that catabolyzes GM1 within lysosomes. Accumulation of GM1 and its asialo form (GA1) occurs primarily in the brain, leading to progressive neurodegeneration and brain dysfunction. Less information is available on the neurochemical pathology in optic nerve and sciatic nerve of GM1- gangliosidosis. Here we analyzed the lipid content and myelin structure in optic and sciatic nerve in 7 and 10 month old normal β-gal (+/?) and GM1-gangliosidosis β-gal (-/-) mice. Optic nerve weight was lower in the β-gal -/- mice than in unaffected β-gal +/? mice, but no difference was seen between the normal and the β-gal -/- mice for sciatic nerve weight. The concentrations of GM1 and GA1 were significantly higher in optic nerve and sciatic nerve in the β-gal -/- mice than in β-gal +/? mice. The content and composition of myelin-enriched cerebrosides, sulfatides, plasmalogen ethanolamines were significantly lower in optic nerve of β-gal -/- mice than in β-gal +/? mice, however cholesteryl esters were enriched in the β-gal -/- mice. No significant abnormalities in these myelin enriched lipids were detected in sciatic nerve of the β-gal -/- mice. The abnormalities in GM1 and myelin lipids in optic nerve of β-gal -/- mice were also associated with abnormalities in the X-ray diffraction pattern including myelin content in fresh nerves [M/(M +B)] and periodicity (d). With the exception of a slight reduction in myelin content, no abnormalities in the X-ray diffraction pattern were observed in sciatic nerve of β-gal -/- mice. The results indicate that neurochemical pathology is greater in optic nerve than in sciatic nerve of β-gal -/- mice. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

by Tsang Yuen Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 98-110). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / CONTENTS --- p.IV / ABBREVIATIONS --- p.VII / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XII / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biochemistry of zinc --- p.1 / Chapter 1.2 --- Zinc within the body --- p.2 / Chapter 1.2.1 --- Roles of zinc in general biochemical processes --- p.4 / Chapter 1.2.2 --- Zinc and zinc-binding proteins (ZnBPs) --- p.9 / Chapter 1.3 --- Zinc in brain --- p.12 / Chapter 1.3.1 --- Distribution of zinc in brain --- p.13 / Chapter 1.3.2 --- Roles of zinc in brain 、 --- p.14 / Chapter 1.4 --- Summary --- p.18 / Chapter 1.5 --- Aim of Study --- p.20 / Chapter 2. --- MATERIALS AND METHODS --- p.22 / Chapter 2.1 --- Detection of zinc binding proteins --- p.22 / Chapter 2.1.1 --- Experimental animal --- p.22 / Chapter 2.1.2 --- Subcellular fractionation of porcine brain --- p.22 / Chapter 2.1.2 --- Determination of protein concentration --- p.24 / Chapter 2.1.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.4 --- Two dimensional polyacrylmide gel electrophoresis (2D-PAGE) --- p.25 / Chapter 2.1.5 --- "Fixing, staining and destaining" --- p.28 / Chapter 2.1.6 --- Western blotting for SDS-PAGE --- p.29 / Chapter 2.1.7 --- 65Zn binding --- p.30 / Chapter 2.1.8 --- Autoradiography --- p.30 / Chapter 2.1.9 --- Data quantification --- p.31 / Chapter 2.2 --- Identification of the 21 kD ZnBP --- p.31 / Chapter 2.2.1 --- N-terminal sequencing of the 21 kD ZnBP --- p.31 / Chapter 2.2.2 --- In situ cyanogen bromide (CNBr) cleavage --- p.32 / Chapter 2.2.3 --- Tricine SDS-PAGE --- p.33 / Chapter 2.2.4 --- Isolation of myelin --- p.34 / Chapter 2.2.5 --- Extraction of the 21 kD ZnBP --- p.35 / Chapter 2.2.6 --- Comparison of the mobility of the 21 kD ZnBP with that of bovine myelin basic protein (MBP) in three different gel electrophoresis system --- p.35 / Chapter 2.3 --- Characterization of the zinc binding properties of MBP --- p.38 / Chapter 2.3.1 --- Concentration-dependence of MBP on 65Zn binding --- p.38 / Chapter 2.3.2 --- Effect of other divalent cations on 65Zn binding to the 21 kD ZnBP --- p.38 / Chapter 2.3.3 --- Effect of pH on 65Zn binding to the 21 kD ZnBP --- p.39 / Chapter 2.3.4 --- Effect of modification of histidine residues in MBP on 65Zn binding --- p.39 / Chapter 2.4 --- Effect of zinc on the interaction between MBP and membrane- lipid --- p.40 / Chapter 2.4.1 --- Effect of zinc on MBP-induced phospholipid vesicle aggregation --- p.40 / Chapter 2.4.2 --- Effect of some divalent cations on MBP-induced phospholipid vesicle aggregation --- p.41 / Chapter 3. --- RESULTS --- p.42 / Chapter 3.1 --- Distribution of ZnBPs in porcine brain --- p.42 / Chapter 3.1.1 --- Subcellular distribution of ZnBPs in porcine brain --- p.42 / Chapter 3.1.2 --- "Comparison of the subcellular distribution of ZnBPs in brain, heart and liver" --- p.45 / Chapter 3.1.3 --- Regional distribution of ZnBPs in porcine brain --- p.48 / Chapter 3.1.4 --- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of total brain homogenates --- p.51 / Chapter 3.2 --- Identification of the 21 kD ZnBP --- p.58 / Chapter 3.2.1 --- N-terminal sequencing of the 21 kD ZnBP --- p.58 / Chapter 3.2.2 --- CNBr cleavage of the 21 kD ZnBP and the N-terminal sequencing of CNBr cleaved fragment --- p.59 / Chapter 3.2.3 --- Detection of the 21 kD ZnBP in myelin fraction and extraction of the 21 kD ZnBP by chloroform-methanol extraction --- p.59 / Chapter 3.2.4 --- Comparison of properties of the 21 kD ZnBP with bovine MBP on three electrophoresis systems --- p.61 / Chapter 3.2.5 --- CNBr cleavage patterns of the 21 kD ZnBP and the bovine MBP --- p.64 / Chapter 3.3 --- Characterization of the zinc binding properties of MBP --- p.69 / Chapter 3.3.1 --- Concentration-dependence of MBP on 65Zn binding --- p.69 / Chapter 3.3.2 --- Effect of other divalent cations on 65Zn binding to the 21 kD ZnBP --- p.69 / Chapter 3.3.3 --- Effect of pH on 65Zn binding to the 21 kD ZnBP --- p.71 / Chapter 3.3.4 --- Effect of modification of histidine residues in MBP on 65Zn binding --- p.73 / Chapter 3.4 --- Effect of zinc on the interaction between MBP and membrane- lipid --- p.76 / Chapter 3.4.1 --- Effect of zinc on MBP-induced phospholipid vesicle aggregation --- p.76 / Chapter 3.4.2 --- Effect of other divalent cations on MBP-induced phospholipid vesicle aggregation --- p.81 / Chapter 4. --- DISCUSSION --- p.83 / Chapter 5. --- CONCLUSIONS --- p.96 / Chapter 6. --- REFERENCES --- p.98

The subject of this thesis is the use of Magnetic Resonance (MR) Imaging to quantify biometric MR indices in the Multiple Sclerosis (MS) fixed post-mortem central nervous system (CNS) tissue. Evaluating these indices in fixed tissue allows for the use of histology to verify the findings of MRI. However, it must first be discovered if the indices can be evaluated in fixed post-mortem spinal cord tissue. There is very little literature in this specific area, though some in the fixed brain, the results of which have been assumed to be equivalent in the spinal cord without proof. Therefore, the methodology must first be verified before the consideration of any index as useful and translatable to in-vivo spinal cord. This thesis concentrates on the evaluation of MR relaxometry methods using the indices T1 and T2 by themselves and to evaluate the myelin content of fixed post-mortem CNS tissue. The Carr-Purcell-Meiboom-Gill (CPMG) and Multicomponent Driven Equilibrium Single Pulse Observation of T1 & T2 (mcDESPOT) sequences are used to calculate T1, T2 and the Myelin Water Fraction (MWF) which is believed to be proportional to myelin content in the CNS. This is performed at 3T in a clinical scanner and at 7T in a small animal and wholebody scanner. The methods are first evaluated for use in fixed post-mortem CNS tissue. The two myelin measurement methods are then compared to histological staining if appropriate and where available to verify that the results obtained are proportional to myelin content. The T1 and T2 values in fixed tissue were found to be shortened in fixed tissue, T2 values were so short as to be at the limits of measurement by a clinical scanner, and values converged in white and grey matter, and therefore contrast was found to be limited between these tissues. Proton density images provided the most contrast between tissues. However, even with shortened T2 values, the CPMG sequence was able to identify the myelin water component in fixed tissue. The mcDESPOT algorithm struggled to separate the myelin water component due to clinical scanner limitations and the shortened, converging T1 and T2 values. However, the mcDESPOT algorithm was successful in discerning the myelin water component in the high signal situation of a small bore 7T peclinical scanner. An evaluation was then made of the usefulness of these indices for translation into clinical imaging. The CPMG sequence was found to be proportional to myelin content under all conditions, and therefore useful for disease monitoring in demyelinating diseases. The mcDESPOT sequence, was found to be proportional to myelin in some conditions, and is likely to be useful for monitoring myelination, though the sequence could not be fully validated in this thesis.

Includes bibliographical references (leaves 194-225) A new method is described that enables longitudinal and cross sections of an individual nerve fibre to be cut at multiple specified sites along the fibre by use of an unique marker system. The method is particularly useful for the correlative study of myelin-axon relationships

Includes bibliographical references (leaves 194-225) / ix, 225, vii leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A new method is described that enables longitudinal and cross sections of an individual nerve fibre to be cut at multiple specified sites along the fibre by use of an unique marker system. The method is particularly useful for the correlative study of myelin-axon relationships / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2002?

T2 relaxation imaging can be used to measure signal from water trapped between myelin bilayers; the ratio of myelin water signal to total water is termed the myelin water fraction (MWF). First, results from multi-component T2 relaxation and diffusion tensor imaging (DTI) were compared for 19 multiple sclerosis (MS) subjects at 1.5 T to better understand how each measure is affected by pathology. In particular, it was determined that the detection of a long-T2 signal within an MS lesion may be indicative of a different underlying pathology than is present in lesions without long-T2 signal. Next, the single-slice T2 relaxation measurement was implemented, refined, and validated at 3.0 T. Scan parameters were varied for phantoms and in-vivo brain, and changes in multi-exponential fit residuals and T2 distribution-derived parameters such as MWF were monitored to determine which scan parameters minimized artifacts. Measurements were compared between 1.5 T and 3.0 T for 10 healthy volunteers. MWF maps were qualitatively similar between field strengths. MWFs were significantly higher at 3.0 T than at 1.5 T, but with a strong correlation between measurements at the different field strengths. Due to long acquisition times, multi-component T2 relaxation has thus far been clinically infeasible. The next study aimed to validate a new 3D multi-component T2 relaxation imaging technique against the 2D single-slice technique most commonly used. Ten healthy volunteers were scanned with both the 2D single-slice and 3D techniques. MWF maps were qualitatively similar between scans. MWF values were highly correlated between the acquisition methods. Although MWF values were generally lower using the 3D technique, they were only significantly so for peripheral brain structures, likely due to increased sensitivity of slab-selective refocusing pulses used for the 3D approach. The 3D T2 relaxation sequence was then applied to the study of MS to take advantage of the increased brain coverage. Thirteen MS subjects and 11 controls underwent T2 relaxation and DTI examinations to produce histograms of MWF and several DTI-derived metrics. MS MWF histograms differed considerably from those of controls, and differences in MS MWF histograms did not mirror differences in DTI histograms relative to matched controls.

The Quaking (qk) gene has been implicated in the development of oligodendroglial cells which are the primary source of myelin in the mammalian central nervous system (CNS). Qk encodes three alternatively spliced variants, QKI-5, QKI-6 and QKI-7, all of which are RNA binding proteins. Loss of QKI-6 and QKI-7 results in a dysmyelination phenotype that is present shortly after birth while loss of QKI-5 results in embryonic lethality. CG4 oligodendroglial cells were transfected with either pIRES2-QKI5 to up regulate QKI-5 expression or a QKI-5 specific siRNA to down regulate QKI-5. Cells were cultured for 6d in differentiation medium (DM) following which total RNA and protein was collected from the cell cultures, and coverslips with attached cells were processed for immunofluorescence. Increased QKI-5 expression following transfection with pIRES2-QKI5 resulted in increased Sirt2 and Plp mRNA expression, but did not affect SIRT2 and PLP protein expression. Down regulation of QKI-5 expression had no significant effect on mRNA or protein levels for QKI-6, QKI-7, Plp or Sirt2. Immunocytochemistry revealed that up regulation of QKI-5 resulted in significantly higher percentage of A2B5+ cells and a lower percentage of GalC+ cells, whereas siRNA treatment resulted in an increase in the percentage of GalC+ cells. Our results suggest QKI-5 regulates oligodendrocyte differentiation and modulates the transcription and availability of target mRNAs, such as Sirt2 and Plp, for translation. In order to gain a more complete understanding of the relationship between qk and both Sirt2 and Plp, future studies would include RNA coimmunoprecipitation, miRNA studies, and expanding the list of target genes to include various cell cycle components.