京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20403号 / 工博第4340号 / 新制||工||1673(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 澤本 光男, 教授 中條 善樹, 教授 赤木 和夫 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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京都大学 / 新制・論文博士 / 博士(工学) / 乙第13542号 / 論工博第4205号 / 新制||工||1984(附属図書館) / (主査)教授 杉野目 道紀, 教授 大江 浩一, 教授 松原 誠二郎 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Pectin has a long history as a food additive. However, elucidation of its fine structural and property relationships remains elusive. Recent research has focused on pectin's ability to complex with divalent heavy metals to aid in characterizing it. Commercial pectins of unknown composition were obtained from local grocers. Purified pectin samples from orange peel, lemon peel, and apple pomace, each of low and high levels of methyl esterification and of unknown distribution pattern were also purchased. Instead of metal complexation, several highly absorbing dyes such as Ruthenium Red, Nile Blue, and Acridine Orange were used to complex with the pectins and their resulting UV-Vis spectral patterns were employed to determine if one can characterize the different pectins. Chemometric methods are also included to aid in distinguishing them apart.

Modification of the lipid A domain of lipopolysaccharide (LPS) is important for the pathogenesis and virulence of various Gram-negative bacteria. The major lipid A species of Helicobacter pylori is significantly different from that of Escherichia coli. H. pylori lipid A contains fewer acyl chains and phosphate groups with only one Kdo sugar attached to the disaccharide backbone. However, H. pylori produces a minor lipid A species that resembles E. coli lipid A, suggesting that the major lipid A species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine (pEtN) transferase, involved in modifying the 1-position of H. pylori lipid A. H. pylori lipid A contains a pEtN unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogens, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. Using in-vitro assay systems, we demonstrate that the modification of the 1-position of H. pylori lipid A is a two-step process involving the removal of the 1-phosphate group by LpxEHP followed by the addition of a pEtN residue catalyzed by EptAHP. As compared to wild-type H. pylori, lpxEHP mutants are extremely sensitive to the cationic peptide polymyxin, thus, demonstrating the importance of modifying the 1-position of lipid A. Furthermore, this work describes another enzyme, YeiU (renamed LpxT), which specifically utilizes the carrier lipid undecaprenyl pyrophsphate (C55-PP) to modify the 1-position of E. coli lipid A. Typically, E. coli lipid A is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'; however, a small fraction contains a diphosphate moiety at the 1-position (lipid A 1-diphosphate). 32P-labeled lipid A obtained from lpxT deficient mutants produces only lipid A, and complementation with a plasmid expressing LpxT restores lipid A 1-diphosphate formation. Inhibition of lipid A 1-diphosphate synthesis was demonstrated by sequestering C55-PP with the cyclic polypeptide antibiotic bacitracin. In conclusion, this work describes two novel pathways for lipid A modification at the 1-position in Gram-negative bacteria.

Cationic steroid antimicrobial agents (CSAs) are a family of bile acid derivatives. These compounds are amphiphilic and mimic endogenous antimicrobial peptides. The antimicrobial activities of CSA-13 have been investigated and due to portent bactericidal activities and low toxicity, a large amount of CSA-13 is demanded for clinic trails and other antimicrobial applications. During our studies, we optimized the synthetic route of CSA-13, so that it can be prepared at the kilogram, even in tons scale. We investigated three routes and one of them is suitable for industry, because only recrystallization is needed in the synthesis. Natural killer T cells (NKT cells) are a kind of lymphocyte that bridge the adaptive immune system with the innate immune system. Once stimulated by glycolipids, NKT cells influence immune responses. To search for better glycolipid ligands, scientists have isolated many natural products to get inspiration. Thrautochysides A-C was isolated from a group of marine protists. These compounds have an interesting structure on their sphingosine lipid chains. We finished the iii synthesis of thraustochyside B, and made substantial progress toward the synthesis of thraustochyside A. Toll like receptors (TLRs) are integral components of the innate immune system. They recognize antigens and induce dendritic cells to give immune responses. TLR1, TLR2 and TLR6 recognize lipopetides, and these TLRs function as heterodimers. TLR1/TLR2 dimer recognition gives inflammatory responses, and TLR2/TLR6 dimer recognition gives immunomodulatory responses. We used modeling of TLRs to find a compound, which can fill the lipid binding pockets of the TLR2 and TLR6 dimer. In our study, we found the peptide chain of the antigen Pam2CSK4 can be replaced by a water soluble polyamine, which confirmed the function of the peptide to increase the water solubility.

Today, paper and paperboard is an important part of the packaging industry, as well as natural part of our daily lives. The alternatives to paper and paperboard is often produced from nonrenewable sources such as petroleum and as demands on sustainability increase in the packaging sector, it gives paper a desirable advantage over plastics. To increase the use of paper, some of the limiting factors for paper needs to be overcome, such as its limited formability. One way to create a paper that can be formed in new ways is to imbibe the paper with an additive that makes its modulus changeable. This would make it possible to control the stiffness of the paper which would make it more formable. This have been the aim of this thesis. To create this formable paper four different approaches were used. Of these four, three included how paper can be treated with polyelectrolytes to be able to increase its modulus when stimuli are applied. The two first approaches were based on the layer-by-layer technique adsorbing alginate and cationic fibrils to form a layer that could be cross-linked, either on the surface of a film (the first approach) or in the network of fibres in a porous paper (the second approach). The last approach was to impregnate the papers with alginate. The results show that the first approach gave a too low adsorption of polyelectrolytes, why no difference could be detected. The second approach resulted in a higher adsorbed amount, but the effects were still too small. The third approach gave a paper which could, depending on the concentration of alginate, either increase or decrease its modulus when cross-linked. The last approach was to create a laminate, using unmodified fibers together with dialcohol cellulose fibres. This resulted in a paper that could be formed using heat and allowing the paper to cool down in the desirable form. In the curriculum for chemistry in Swedish high schools there is a paragraph that says that research in chemistry should be a part of the content the students learn. This is a challenge both to teachers and researchers. For teachers, to find research at an appropriate level and teach it in an understandable way. For researchers, to communicate the research so it is understandable for society. With this as a motivation, a second part of this thesis discuss the method flipped classroom in chemistry education at Swedish high schools. It was seen that from the teacher’s perspective, flipped classroom meant advantages that could be categorized into five categories. The categories that was found were Time to interact, Student responsibility, dialogue, laboratory work and understanding and using the language. / Pappers- och kartongindustrin är idag en viktig del av förpackningsindustrin. Alternativ till kartong är ofta producerat av icke-förnybara råvaror och idag när kraven på hållbarhet ökar ger detta kartong en fördel mot plast. För att öka användningsområdena för kartong så måste de begränsningar som materialet har idag övervinnas. Detta gäller till exempel formbarheten. Ett sätt att skapa kartong som kan bli format på nya sätt är att göra en additiv tillsats till papperet som har egenskapen att det kan variera sin styvhet. Detta skulle göra det möjligt att kontrollera papperets styvhet, vilket har varit målet för det här arbetet. Genom att använda fyra olika arbetssätt har denna egenskap försökts åstadkommas. Tre av arbetssätten har behandlat papper med polyelektrolyter vars E-modul kan ökas med hjälp av något stimuli. I de två första försöken applicerades polyelektrolyterna genom den så kallade lager-på-lager-tekniken, där alginat och katjoniska cellulosafibriller adsorberades, i första försöket på ytan av en film och i det andra på ytan av fibrerna i fibernätverket i ett poröst papper. I det tredje försöket impregnerades de orösa papperna med alginat. Resultatet från det första försöket var att adsorptionen inte gav någon effekt eftersom den absorberande mängden var för låg. I det andra försöket var adsorptionen högre men det påverkade inte de mekaniska egenskaperna nämnvärt. I det tredje försöket med impregnering så kunde en ändring av E-modulen observeras när systemet tvärbands. I det sista försöket användes en metod som byggde på laminering. Ett ark av modifierade fibrer guskades ihop med ett ark med dialkoholcellulosefibrer. De försök som gjordes tyder på att papperet kunde formas och kunde fås att behålla formen om det värmdes och kyldes i den önskade formen. I läroplanen för kemi på gymnasiet finns en paragraf som beskriver hur kemiundervisningen ska ha inslag av forskning. Detta är en utmaning för både lärare och forskare. Lärarnas utmaning består i att finna forskning på en bra nivå för eleverna och att förklara den på ett förståeligt sätt. För forskare består utmaningen i att kommunicera forskningen på ett förståeligt sätt till samhället. Med detta som motivering har detta arbete en utbildningsdel. I den har lärares perspektiv på metoden ”flipped classroom” i kemiklassrummet undersökts. Flipped classroom beskrevs ha styrkor och dessa kunde kategoriseras i fem kategorier. Kategorierna var tid för interaktion, elevens ansvar, dialog, laborationer och förstå och använda språket.

Arginine transport is primarily mediated by the cationic amino acid transporters (CATs) in mammalian cells, but in aves the y+, b0,+ and B0,+ transport systems have also been observed. Arginine is the limiting catabolic substrate required for the production of nitric oxide (NO), a highly reactive compound that acts as a signaling molecule or killing compound. NO is synthesized by inducible nitric oxide synthase (iNOS) by macrophages for pathogen clearance. In mammals, CAT-2B is responsible for ARG import in the macrophage for NO synthesis, but the chicken CAT-2B isoform does not transport ARG. Therefore the objective of these studies was to identify the CAT(s) involved in mediating ARG uptake during a NO response in the chicken macrophage. Experiments were performed to measure: 1) ARG transporter mRNA and NO production from three sources of macrophages (HD11 cell line, n=6; primary 32d Cobb 500, n=8; Hyline W36, n=7) in response to Escherichia coli lipopolysaccharide (LPS); 2) the effect of CAT over-expression on NO production in response to LPS (HD11 cell line; n=8). In response to LPS iNOS mRNA abundance increased (P<0.05) 8.5-fold in the HD11 macrophages, 3.22-fold in broiler macrophages and 2.79-fold in layer macrophages. In all cells, CAT-1 was induced and CAT-2A increased (P<0.05) between 1.28 and 1.68-fold. CAT-2B was not detected at any time point or treatment condition. In the virally transformed chicken macrophage cell line (HD11) CAT-3 mRNA was induced, but in primary cells CAT-3 increased (P<0.05) 1.27-fold in broilers and 1.23-fold in layers. Transiently transfected chicken macrophages produce NO independent of LPS treatment by 6h, mock transfected controls did not respond by 6h. In the presence of LPS, CAT-1 transfected macrophages produced 50.0% more NO than mock transfected cells (P<0.05). CAT-2A and CAT-3 transfected macrophages produced only 17.6% and 72.1% of the total NO produced by controls (P<0.05). These results indicate that CAT-1 and CAT-3 are both sufficient to sustain ARG import for NO production in the chicken macrophage, but that CAT-1 produces a maximal response. These results also show that iNOS, despite its name, is constitutively present and can be activated by induction of CATs to import ARG.

Current treatment for posterior segment ocular diseases requires intravitreal injections administered every 4-6 weeks. The potential for siRNA to be used to treat these diseases is extremely attractive due to the specificity of these molecules and their potential for making long term changes to the expression patterns of the cells. Due to physiological recognition, however siRNA undergoes rapid degradation upon application. The development of cationic nanogels using polymeric “smart” biomaterials with degradable components to transport siRNA is described. pH – sensitive N, N dimethylaminoethyl methacrylate (DMAEMA) was crosslinked with thermo-sensitive diethylene glycol methacrylate (DEGMA), by free radical emulsion-precipitation polymerization. Size, charge and morphology were analyzed to assess potential as a nanovehicle. Through modification of the particle composition, cationic nanogels, determined by zeta potential, with sizes of approximately 160 nm confirmed with dynamic light scattering (DLS), were synthesized. A composition of 55:45 (DEGMA:DMAEMA); a size and charge ideal for cellular uptake. These particles had minimal impact on cell proliferation and exhibited spherical morphology when imaged by TEM at physiological pH. The structure was maintained between pH 3.5-9. Sensitivity to pH was shown by DLS through swelling at physiological pH, which may be useful can be taken advantage of in future studies for loading and release. Degradation with a reducing agent was shown using gel permeation chromatography, DLS and turbidity analysis. The results suggest this formula will undergo degradation in the cell. Reducing environments mimicking intracellular conditions that promoted degradation of the crosslinker showed enhanced release of dexamethasone phosphate as a model drug. Ongoing work is focused on examining gene silencing using these formulations. / Thesis / Master of Applied Science (MASc)