Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / Includes bibliographical references (p. 149-160). Also available on the Internet.

Orientador: Rosicleire Veríssimo Silveira / Resumo: O objetivo deste trabalho a incorporação de ácidos graxos essenciais nas membranas dos embriões de Astyanax altiparanae, visando a determinação de protocolos para a crioconservação de embriões desta espécie de peixe neotropical.Para realização do experimento foram selecionados 2400 espécimes deA. altiparanae. Um grupo foi alimentado com ração comum, denominado de Ração Comercial (RC), e outro alimentado com ração comum acrescida de 5% de óleo de peixe marinho, denominado de Ração Comercial com Óleo de peixe marinho (RC+O). No Capítulo I, foi realizado análise de ácidos graxos no fígado, gônada e embrião. E verificado a mobilização destes ácidos graxos e sua influência. No Capítulo IIverificamos a permeabilização do córion nos embriões quando expostos a solução de pronase-E 25mg/mL, por períodos de 10, 20, 30s. Também foi analisado a sensibilidade dos embriões RC e RC+O aos crioprotetores. Os embriões foram expostos a soluções de propileno glicol, metanol, dimetil sulfóxido, dimetil, formamida, dimetil acetamida e glicerol, por 20 minutos. Verificou-se a taxa de eclosão e viabilidade das larvas após a eclosão. No Capítulo III os embriões do grupo RC e RC+O foram vitrificados em solução de propileno glicol 5M, na fase de seis somitos.No capítulo IV buscou-se o resfriamento dos embriões de RC e RC+O a tempeturas de 15°C, 5°C e 0°C, por um período de 12h. Os embriões foram expostos a soluções sem crioprotetor, com propileno glicol 1M e 2M. / Abstract: The objective of this work was the incorporation of essential fatty acids in the membranes of the embryos of Astyanax altiparanae, aiming the determination of protocols for the cryopreservation of embryos of this neotropical fish species. For the experiment, 2400 specimens of A. altiparanae were selected. One group was fed with common ration, called Commercial Ration (CR), and the other was fed with common ration plus 5% of marine fish oil, called Commercial Ration with Marine Fish Oil (CR + O). In Chapter I, analysis of fatty acids in the liver, gonad and embryo was performed. And we verified the mobilization of these fatty acids and their influence. In Chapter II we verified the permeabilization of the chorion in the embryos when exposed to 25mg / mL pronase-E solution, for periods of 10, 20, 30s. The sensitivity of the CR and CR + O embryos to the cryoprotectants was also analyzed. The embryos were exposed to solutions of propylene glycol, methanol, dimethyl sulfoxide, dimethyl, formamide, dimethyl acetamide and glycerol for 20 minutes. The hatch rate and viability of the larvae after hatching were checked. In Chapter III the embryos of the CR and CR + O group were vitrified in 5M propylene glycol solution in the six somites phase. In Chapter IV, the cooling of the CR and CR + O embryos was sought at temperatures of 15 ° C, 5 ° C and 0 ° C for a period of 12 hours. The embryos were exposed to cryoprotectant solutions with 1M and 2M propylene glycol. / Doutor

Drosophila are used extensively for genetic, developmental and now molecular biology research. At present, germline transformation of these organisms can only be achieved by microinjection of P-element vectors into the pole cells of young embryos. The technique of microinjection however, requires a delicate touch and is quite laborious. Therefore, the development of a rapid and simple technique was investigated. Electroporation, like microinjection, is a physical means of introducing DNA into a cell and is therefore potentially applicable to all cell types. Electroporation involves the use of an electrical current to create pores in the membrane of a cell. Macromolecules, such as DNA may enter a cell via these pores. Electroporation is a quick, reproducible, and efficient method for transforming cells. Through studies of the survival and permeability of Drosophila melanogaster embryos exposed to electrical currents, it was discovered that although the survival of the embryos decreased steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. Few embryos survived this extreme voltage required for dye uptake. Attempts to introduce DNA into dechorionated Drosophila embryos utilizing this technique however, produced no transformants. These results suggested that the remaining protective coatings of the dechorionated embryo were obstructing efficient pore formation, thus preventing DNA penetration. In view of these results, methods to eliminate the wax layer, present between the chorion and vitelline membrane of laid eggs, were examined. Wax removal by detergent solubilization, solvent extraction and melting by heating were investigated, yet did not produce a satisfactory procedure. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Cumulative clinical and experimental evidence indicates that volatile sulphur compounds (VSC) the principal components of oral malodour, may play an important role in the pathogenesis of periodontal disease. As their (H₂S and CH₃SH) concentrations in gingival sulci increase with the severity of periodontal involvement, the objective of this investigation is to ascertain if they exert an effect on the permeability of oral mucosa. Permeability determinations were performed on excised porcine sublingual mucosal specimens which consisted of non-keratinized epithelium, basal membrane and connective tissue layers mounted in a two compartment perfusion apparatus. Using radioactive and fluorescent-labelled penetrants, it was found that exposure of the epithelial surface to an atmosphere containing physiological concentrations of both thiols (15 ng H₂S or CH₃SH / ml of 95% air - 5% C0₂) increased the permeability of the mucosa to (³⁵S)-S0₄⁻², (³H)-prostaglandin E₂ (PGE₂) and fluorescein isothiocyanate labelled E. coli lipopolysaccharide (F-LPS). A three hour exposure of the mucosa to H₂S and CH₃SH resulted in a 75% and 103% increase respectively in permeability to (³⁵S)-labelled sulphate ion. Similarly, the mercaptan induced up to a 70% increase in permeability of the mucosa to (³H)-prostaglandin E₂. The magnitude of changes in the permeability were found to depend on duration of exposure to the thiols and to their concentration. Studies using (³⁵S)-H₂S suggest that the observed changes in the tissue permeability are related to the reaction of the thiols with tissue components. In addition, the (³⁵S)-H₂S is capable of perfusing through all three layers of the mucosa at 12.3 ng / cm². In contrast to H₂S , the CH₃SH effect was irreversible in control air / C0₂ environment. This infers that CH₃SH is potentially a more deleterious agent to the tissue barrier. However, its effect can also be reversed by treatment of tissues with 0.22% ZnCl₂ either prior to or after exposure to mercaptan. This suggests that Zn⁺² ion may be useful in preventing the potentially harmful effects of VSC. Fluorescent studies with F-LPS indicate that thiols can also potentiate the penetration of endotoxin. Whereas the fluorescence of the F-LPS in control systems was confined to the superficial epithelial layer in contact with the endotoxin, the CH₃SH- exposed mucosa exhibited fluorescence throughout the epithelial and connective tissue layers. Fluorescent staining of the mucosal specimens with fluorescein diacetate followed by counter staining with ethidium bromide provides evidence of membrane impairment to some cells by CH₃SH. Collectively these observations provide strong experimental evidence that the VSC, products of putrefaction produced in the gingival sulcus by oral microflora, may adversely affect the integrity of the crevicular barrier to deleterious agents and thus contribute to the etiology of periodontal disease. / Dentistry, Faculty of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21958号 / 医博第4500号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 松原 和夫, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Membrane traffic in polarised cells was investigated by growing Madin-Darby canine kidney (MOCK) cells on ·permeable polycarbonate filter supports which allowed access to both sides of the cell monolayer. Membrane glycoconjugates on the apical and basolateral cell surfaces were labelled enzymatically with ³H- and ¹⁴C-galactose, respectively, to provide covalent membrane markers. Experiments were done to quantitate membrane traffic during endocytosis at the respective plasma membrane domains and that due to transcytosis. Internalized label was quantitatively distinguished from label on the respective cell surface by its resistance to removal by glycosidases.

The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared pawwithdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.

Measuring transmembrane water exchange can provide potential biomarkers for tumors and brain disorders. Diffusion Magnetic Resonance Imaging (dMRI) is a well-established tool that can non-invasively measure water exchange across cell membranes. Diffusion Exchange Spectroscopy (DEXSY) is one of the dMRI-based frameworks used to estimate exchange. DEXSY provides a detailed picture of multi-site exchange processes but requires a large quantity of data. Several models based on the DEXSY framework have been proposed to reduce the acquisition time. Filter Exchange Imaging (FEXI) and curvature models are two of them that only require certain samples of the DEXSY dataset. Diffusion-Exchange Weighted (DEW) Imaging model is another data reduction method accounting for restricted diffusion within cells and can use a specific subset of the DEXSY dataset to measure exchange. Furthermore, a more general expression of the DEXSY signal, referred to as the general model, can theoretically analyze the full space or reduced DEXSY datasets and estimate exchange. However, the results of the subsampling schemes and the data reduction models have not been compared to the full space estimation.  Therefore, this thesis aims to experimentally explore the feasibility of estimating exchange using these four models (the general, FEXI, curvature and DEW models) with the data acquired using a low-field benchtop MR scanner, and compare the estimates from the general model with different subsampling schemes and the data reduction models to the full space estimation. For this purpose, a double diffusion encoding (DDE) sequence was modified from an existing sequence on the benchtop MR scanner and a DEXSY experiment was conducted on this MR scanner and a yeast phantom to acquire a full space dataset. The exchange parameters estimated from the full space dataset using the general model were used as "ground truths" to evaluate the estimates from the reduced datasets analyzed using the general, FEXI and curvature models. Moreover, two alternative subsampling schemes named the shifted DEW and new trajectory schemes were proposed and employed to measure exchange. The results indicate that all the methods except the curvature sampling scheme employed with both the general and curvature models provided comparable estimates to the "ground truths". The shifted DEW and new trajectory sampling schemes performed better over others in terms of consistency with the "ground truths" and low variations between voxels, suggesting the theoretical and experimental optimization of these two subsampling schemes can be further studied and developed.

No description available.

No description available.