Indiana University-Purdue University Indianapolis (IUPUI) / A variety of polymers of synthetic origins (e.g., poly(ethylene glycol) or PEG) and naturally derived macromolecules (e.g., silk fibroin or gelatin) have been explored as the backbone materials for hydrogel crosslinking. Purely synthetic hydrogels are usually inert, covalently crosslinked, and have limited degradability unless degradable macromers are synthesized and incorporated into the hydrogel network. Conversely, naturally derived macromers often contain bioactive motifs that can provide biomimicry to the resulting hydrogels. However, hydrogels fabricated from a single macromer often have limitations inherent to the macromer itself. For example, to obtain high modulus PEG-based hydrogels requires an increase in macromer and crosslinker content. This is associated with an increase in radical concentration during polymerization which may cause death of encapsulated cells. Pure gelatin (G) hydrogels have weak mechanical properties and gelatin undergoes thermo-reversible physical gelation. Covalent crosslinking is usually necessary to produce stable gelatin hydrogels, particularly at physiological temperatures. The limitations of these hydrogels may be circumvented by combining them with another macromer (e.g., silk fibroin) to form hybrid hydrogels. Silk fibroin (SF) from Bombyx mori silkworms offers high mechanical strength, slow enzymatic degradability, and can easily form physical hydrogels. The first objective of this thesis was to evaluate the effect of sonication and the presence of synthetic polymer (e.g., poly (ethylene glycol) diacrylate or PEGDA) or natural macromer (e.g., gelatin) on SF physical gelation kinetics. SF physical gelation was assessed qualitatively via tilt tests. Gelation of pure SF solutions was compared to mixtures of SF and PEGDA or G, both with or without sonication of SF prior to mixing. The effect of gelatin on SF gelation was also evaluated quantitatively via real time in situ rheometry. Sonication accelerated gelation of SF from days to hours or minutes depending on SF concentration and sonication intensity. Both PEGDA and G were shown to accelerate SF physical gelation when added to SF and sonicated SF (SSF) solutions. The second objective was to develop a simple strategy to modulate covalently crosslinked PEG-based hydrogel properties by physically entrapping silk fibroin. The physical entrapment of silk fibroin provides an alternative method to increase gel storage modulus (G’) without the cytotoxic effect of increasing macromer and crosslinker concentration, or altering degradation kinetics by increasing co-monomer concentration. The effect of SF entrapment on gel physical and mechanical properties, as well as hydrolytic degradation and chemical gelation kinetics were characterized. SF physical crosslinking within the PEG-based network was shown to increase gel storage moduli by two days after gel fabrication. There was no change hydrolytic degradation rate associated with the increased moduli. SF entrapment did not affect gelation efficiency, but did alter gel physical properties. The third objective of this thesis was to develop a silk-gelatin in situ forming hybrid hydrogel for affinity-based growth factor sequestration and release and in vitro cell culture. SF provides mechanical strength and stability, whereas G contains bioactive motifs that can provide biomimicry to the gel network. Hydrogel G’ and its dependency on temperature, SF processing conditions, and secondary in situ chemical crosslinking (i.e., genipin crosslinking) were studied. Gelatin can be conjugated with heparin, a glycosaminoglycan, to impart growth factor (GF) binding affinity. Growth factor sequestration and release were evaluated in a pair of designed experiments. The hybrid gels were evaluated as substrates for human mesenchymal stem cell proliferation.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12821号 / 論農博第2794号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4816(農学部図書室) / 31308 / (主査)教授 河田 照雄, 教授 保川 清, 准教授 橋本 渉 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21115号 / 工博第4479号 / 新制||工||1696(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 辻 康之, 教授 近藤 輝幸, 教授 中村 正治 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Tyrophagus putrescentiae (Schrank) (Sarcoptiformes: Acaridae), also known as the ham mite, may infest dry cured hams during the aging process. The fumigant methyl bromide is currently used to control mite infestations, but eventually will not be available for use since it contributes to the depletion of the ozone layer. The use of ham nets treated with xanthan gum, carrageenan, propylene glycol alginate, propylene glycol (PG), and lard were evaluated for their impact on mite orientation to or oviposition on treated or untreated ham cubes, mite reproduction and population growth over a 10-week period. When nets were infused with gum and PG, behavioral tests indicated that greater than 95% of the mites oriented to the ham cubes that were wrapped in untreated nets when compared to treated nets and no eggs were laid on the latter. The reproduction assays indicated that there were fewer (P < 0.05) T. putrescentiae produced over a two-week period on ham cubes covered with both gum and PG treated nets when compared to the untreated or gum-only treated nets over the 10-week storage period of the experiment. Medium and high concentrations of PG treatments had the lowest mite reproduction rates. No more than four mites could be found on each of these treatments in comparison to 200-300 mites that were on the untreated ham cubes. When nets were infused with gum, PG, and lard, behavioral tests indicated that fewer mites oriented to the ham cubes that were wrapped with gum, lard, and medium PG than those with untreated nets. The oviposition assays revealed that on average less than three eggs were laid on the ham cubes with treated nets in comparison to 69-165 eggs on the untreated ham cubes. Reproduction assays demonstrated that fewer T. putrescentiae (P < 0.05) were on ham cubes with treated nets containing PG when compared to the number of mites on ham cubes with untreated nets over 10 weeks of storage. Lard infused nets without PG did not decrease the mite population (P > 0.05). The net without coating slowed the growth and reproduction of T. putrescentiae since net controls had fewer mites (P < 0.05) than controls without nets. With a few exceptions, fungi were not present on ham cubes that were treated with PG-containing nets over 10 weeks of storage. This research demonstrated the efficacy of using nets treated with food-grade ingredients during ham aging to control mite infestations on a laboratory scale. Further research will be conducted to determine the effectiveness of the same treated nets on whole hams in commercial aging rooms.

The oncogene c-MYC has guanine-rich and complementary cytosine-rich sequences in its P1 promoter region. The P1 promoter is responsible for over 90% of the c-MYC expression. Downregulation of c-MYC expression represents a novel therapeutic approach to more than 50% of all cancers. A stable i-motif formed by the c-MYC C-rich sequence would be an attractive target for cancer treatment. We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5-5.5). The question is whether c-MYC promoter sequence i-motif will be stable at physiological pH. In this work, we have investigated the stability of mutant c-MYC i-motif in solutions having pH values from 4 to 7 and containing co-solutes or molecular crowding agents. The crowded nuclear environment was modeled by the addition of polyethylene glycol (PEG, having molecular weights from 200 to 12000 g/mol) at concentrations of 10% to 40% w/w. Circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC) were used to establish the presence and stability of c-MYC i-motifs in buffer solutions having pH values of 4 to 7. The results of these studies are: 1) the addition of up to 20% w/w glycerol does not increase i-motif stability, 2) the addition of 30% PEG results in an increase in i-motif stability to pH values as high as 6.7, 3) i-motif stability is increased with increased PEG concentration and increased PEG molecular weight, and 4) the effects of PEG size and concentration are not linear, with larger PEGs forming DNA/PEG complexes, which destabilize the i-motif. In summary, we have shown that the c-MYC i-motif can exist as a stable structure at pH as high as 6.7 in a crowded environment. Molecular crowding, largely an excluded volume effect, drives the formation of the more compact i-motif, even at higher pH values where the cytosine imino-nitrogen is deprotonated and neutral C-C pairs can form only two H-bonds. Based on this research, it seems possible that a stable c-MYC promoter sequence i-motif could form at physiological pH and would be a reasonable drug target for new cancer therapies.

No description available.

In this thesis, the successful fabrication of 3D-printed microfluidic devices will be discussed. Fabrication is performed with a low-cost commercially available stereolithographic 3D printer utilizing a custom PEGDA resin formulation tailored for low non-specific protein adsorption based on my colleagues' work [Rogers et al., Anal. Chem. 83, 6418 (2011)]. Horizontal microfluidic channels with designed rectangular cross sectional dimensions as small as 300 um wide and 150 um tall are printed with 100% yield, as are cylindrical vertical microfluidic channels with 300 um designed (334 um actual) diameters. Moreover, two different resins developed by our group are utilized in the process of 3D-printing which is the novel aspect about this thesis since other groups have not done research on this aspect of 3D-printing.

Amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(vinylidene fluoride) (PVDF) were synthesized by RAFT polymerization. The commercial poly(ethylene glycol) methyl ether (Me-PEG-OH, 20 Kg/mol) and difunctional polyethylene glycol (OH-PEG-OH, 20 Kg/mol) were used to synthesize diblock copolymers (Me-PEG-b-PVDF), and triblock copolymers (PVDF-b-PEG-b-PVDF) respectively. For the synthesis, the esterification reaction followed by the SN2 reaction was employed to make macro CTA (Me-PEG-XA, XA-PEG-XA, XA refers to the xanthate group). The macro CTAs were used further for VDF polymerization in dimethyl carbonate (DMC) inside the autoclave. Different molecular weights of the PVDF block (whether in the diblocks or the tribolcks) were obtained based on changes in the reaction time. The resulting block copolymers were molecularly characterized by FT-IR, 1H,19F-NMR, and SEC. The thermal properties were studied by DSC and TGA. Furthermore, the crystalline phase characterization was investigated by XRD and FT-IR. Being the obtained block copolymers are amphiphilic, their self-assembly was achieved by nanoprecipitation in DMF/water, and they were analyzed by DLS and TEM.

This dissertation focuses on improving the chromatographic efficiency of polymeric organic monoliths by characterizing and optimizing the bed morphology. In-situ characterization techniques such as capillary flow porometry (CFP), 3-dimensional scanning electron microscopy (3D SEM) and conductivity measurements were developed and implemented to quantitatively characterize the morphology of poly(ethylene glycol) diacrylate (PEGDA) monoliths. The CFP measurements for monoliths prepared by the same procedure in capillaries with different diameters (i.e., 75, 150, and 250 μm) clearly showed a change in average through-pore size with capillary diameter, thus, certifying the need for in-situ measurement techniques. Serial sectioning and imaging of PEGDA monoliths using 3D SEM gave quantitative information about the average pore size, porosity, radial heterogeneity and tortuosity of the monolith. Chromatographic efficiency was better for a monolith with smaller average pore size (i.e., 5.23 μm), porosity (i.e., 0.49), radial heterogeneity (i.e., 0.20) and tortuosity (i.e., 1.50) compared to another monolith with values of 5.90 μm, 0.59, 0.50 and 2.34, respectively. Other than providing information about monolith morphology, these techniques also aided in identifying factors governing morphological changes, such as capillary diameter, polymerization method, physical/chemical properties of the pre-polymer constituents and weight proportion of the same. A statistical model was developed for optimizing the weight proportion of pre-polymer constituents from their physical/chemical properties for improved chromatographic efficiency. Fabricated PEGDA columns were used for liquid chromatography of small molecules such as phenols, hydroxyl benzoic acids, and alkyl parabens. The chromatographic retention mechanism was determined to be principally reversed-phase (RP) with additional hydrogen bonding between the polar groups of the analytes and the ethylene oxide groups embedded in the monolith structure. The chromatographic efficiency measured for a non-retained compound (uracil) was 186,000 plates/m when corrected for injector dead volume. High resolution gradient separations of selected pharmaceutical compounds and phenylurea herbicides were achieved in less than 18 min. Column preparation was highly reproducible, with relative standard deviation (RSD) values less than 2.1%, based on retention times of the phenol standards (3 different columns). A further improvement in chromatographic performance was achieved for monoliths fabricated using a different polymerization method, i.e., living free-radical polymerization (LFRP). The columns gave an unprecedented column performance of 238, 000 plates/m for a non-retained compound under RP conditions.

Climate change and its effects on our society is a steadily growing problem. In 2010, the industry sector accounted for more than 30% of the global greenhouse gas emissions. The chemical industry is one of the industrial subsectors responsible for the highest emissions of greenhouse gas. To reach the climate goals it is therefore urgent to find more sustainable options for production of chemicals in general. Synthetic biology and microbial cell factories are growing fields that have received much attention for inferring promising sustainable alternative production routes for various compounds. When it comes to microbial cell factories, cyanobacteria infer many advantages over heterotrophs. Cyanobacteria can for instance convert atmospheric CO2 into valuable compounds through photosynthesis using the light reaction and the Calvin-Benson cycle. In the present work, the freshwater cyanobacterium Synechocystis sp. PCC 6803 is metabolically engineered to produce 1,2-propanediol; an important chemical feedstock for which there is a great interest in finding a sustainable production route as an alternative to the current petrochemical one. Seven different constructs are designed for introduction and expression of a three-step heterologous metabolic pathway for 1,2-propanediol production. Two strains of Synechocystis are successfully engineered, with the heterologous pathway chromosomally integrated at the Neutral Site I through homologous recombination with an integrative plasmid targeting this genomic site. One of the three heterologous genes (mgsA) of the pathway was successfully translated as shown in a Western immunoblot. In a SDS-PAGE a band of 40 kDa was detected, corresponding to the size of both the sADH and YqhD enzymes.