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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24794号 / 医博第4986号 / 新制||医||1066(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 寺田 智祐, 教授 武藤 学, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

With the growing concern of the accumulation of plastic-based food packaging waste, the search for bio-based biodegradable packages is on the rise. These materials differ from their petro-based counterparts in their degradation rates, which are much higher in the former. Not only do bio-based biodegradable materials degrade faster during post-usage processes but also they age faster during usage and storage, which affects their performance and functionality. The application of noninvasive testing methods with the capability to report on the matrix’s state could assist in the development of a more ubiquitous way to assess ageing in food packaging, particularly in biodegradable ones. To this end, the performance of a luminescence spectroscopy technique based on three luminescent probes, one intrinsic to the matrix and two added, was monitored and the sensitivity of the probes to report on ageing was analyzed. Biodegradable films were made of 2% gelatin (type A) and 0.5% glycerol (plasticizer). Gelatin contains an intrinsic fluorophore, the aromatic amino acid tyrosine (Tyr), which can report on the molecular mobility of a matrix. Additionally, the films were doped with two extrinsic fluorophores, Fast Green FCF (FG) at 0.124 mM and pyranine (Pyr) at 0.05 mM, which can report on the physical state and available free water within a matrix, respectively. Films were casted onto plastic Petri dishes and stored at five relative humidities (RHs), namely 2.5, 25, 33, 53, and 75%, for five weeks with measurement collection every week. Films were tested using fluorescence spectroscopy at excitation and emission range wavelengths optimized depending on the assessed probe. Additional measurements to determine moisture content, changes in secondary protein structure using FTIR spectroscopy, and mechanical properties using a Universal Testing Machine in tensile mode aided in the evaluation of the sensitivity of the luminescent probes in sensing ageing. Luminescent probes, intrinsic or added, have the capability to assess the physical state of the films in situ and can provide molecular level sensing of their local environment. Tyr emission showed a sharp increase in fluorescence intensity in films stored at low RH as a function of time. FG showed a similar pattern to that of Tyr but higher sensitivity to changes along the observed period. The two characteristics emission bands of Pyr provide information on the state of water within the matrix. Although the results on this probe hinted microstructural rearrangements within the films as a function of time, the sensitivity of this probe was not high enough at the conditions evaluated and provided limited information on films’ solvation. The sensitivity of the luminescent probes to changes during ageing were revealed through correlation of the photophysical properties of the two effective probes, Tyr and FG, and the mechanical properties of the films at different RH through storage. Both methods, mechanical and optical, were similarly sensitive to changes during ageing particularly after 3-week storage. However,, it can be speculated that because of the different scales at which optical and mechanical measurements report (local vs. bulk), the methods, they could also complement each other. These findings suggest that, in principle, a luminescence spectroscopy technique using intrinsic and extrinsic probes can replace mechanical testing to noninvasively monitor structural changes and stability of biodegradable packaging as a function of time.

No description available.

Food insecurity is a growing issue in the United States. Iron deficiency is the most common form of nutritional deficiency in patients with endothelial dysfunction and vascular-related diseases. This preliminary study lays the groundwork for the “Nutrient deficiency-on-a-chip” model. Endothelial cells are cultured on mechanically tunable, enzymatically cross-linked gelatin and treated with deferoxamine, an iron chelator, or angiotensin II were used to simulate a nutrient deficient and diseased environment, respectively. As oxidative stress and disturbed barrier function are the most prevailing mechanism of angiotensin II and iron deficiency induced endothelial dysfunction, to test our model we investigated the changes in reactive oxygen species production and VE-cadherin expression in engineered endothelium. Both angiotensin II and deferoxamine treated engineered endothelium showed an increase in oxidative stress and disturbed barrier function. This in vitro model can be a useful tool to better understand disease mechanisms associated with nutrient deficiency and identify novel therapeutics.

This research aims to develop a tissue-mimicking material and produce a 3D gelatin that has density of approximately a human lung, which is in the ranges of (0.25 – 0.35) g/cm3. Tissue equivalent models are important in order to study the radiation dose planned for patients. To achieve the desired density of a human tissue, different types of gelatin were whisked for 300 seconds using a typical hand mixer. The mechanical properties of the gelatin mixtures, standard and foamed, were evaluated by applying different forces. The mechanical properties for the gels were measured using an indentation technique, which showed that the gels act as elastic materials. The mechanical properties of the foams were also evaluated. Mixtures that contained 300 bloom gelatin, glycerol, and sorbitol, were whisked for 60, 180, 300 seconds to achieve different densities evaluated by CT imaging. The density of the180 - and 300 - seconds gelatin foams were found to be 0.33 ± 0.16 and 0.33  0.052 g/cm3, respectively, which is similar to the human lung density. Finally, FXO gel sheets and the FXO foam sheets were irradiated and the radiosensitivity quantified by measuring transmission using a spectrometer. The change in the attenuation coefficient was linear with dose. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25188号 / 医博第5074号 / 新制||医||1072(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 永井 純正, 教授 椛島 健治, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / As liver disease becomes more prevalent, the development of an in vitro culture system to study disease progression and its repair mechanisms is essential. Typically, 2D cultures are used to investigate liver cell (e.g., hepatocyte) function in vitro; however, hepatocytes lose function rapidly when they were isolated from the liver. This has promoted researchers to develop 3D scaffolds to recreate the natural microenvironment of hepatic cells. For example, gelatin-based hydrogels have been increasingly used to promote cell fate processes in 3D. Most gelatin-based systems require the use of physical gelation or non-specific chemical crosslinking. Both of these methods yield gelatin hydrogels with highly interdependent material properties (e.g., bioactivity and matrix stiffness). The purpose of this thesis research was to prepare modularly crosslinked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatic cell fate in 3D. The first objective was to establish tunable gelatin-based thiol-norbornene hydrogels and to demonstrate that the mechanical and biological properties of gelatin hydrogels can be independently adjusted. Furthermore, norbornene and heparin dual-functionalized gelatin (i.e., GelNB-Hep) was prepared and used to sequester and slowly release hepatocyte growth factor (HGF). The second objective was to investigate the viability and functions of hepatocytes encapsulated in gelatin-based hydrogels. Hepatocellular carcinoma cells, Huh7, were used as a model cell type to demonstrate the cytocompatibility of the system. The properties of GelNB hydrogels were modularly tuned to systematically evaluate the effects of matrix properties on cell viability and functions, including CYP3A4 activity and urea secretion. The last objective was to examine the effect of heparin immobilization on hepatocyte viability and functions. The conjugation of heparin onto GelNB led to suppressed Huh7 cell metabolic activity and improved hepatocellular functions. This hybrid hydrogel system should provide a promising 3D cell culture platform for studying cell fate processes.

Innovativa teknologier, produkter och processer krävs på grund av marknadens ökande behov av bioprodukter med låga koldioxidavtryck. Idén att producera flyktiga fettsyror via biologiska processer, främst genom anaerob fermentering med blandkulturer, har återupplivats på grund av den omfattande exploateringen av oljeresurser. Målet för denna studie var att utvärdera kvaliteten och livskraften för produktion av flyktiga fettsyror från enkla substrat. Flyktiga fettsyror-generering undersöktes i ett satsvis syrafermenteringssystem för tre enkla substrat (olja, stärkelse, och gelatin). Vi visade att ett initialt surt pH var fördelaktigt för acidogenes och den totala genereringen av flyktiga fettsyror för alla tre substraten. Den högsta produktionseffektiviteten av flyktiga fettsyror uppnåddes och flyktiga fettsyror genomgick evolutionär utveckling med blandad kulturfermentering som kördes vid ett initialt surt pH på 5,0. 7933,08 mg COD/L smörsyra producerades från stärkelse vid pH 3,87 på dag 15, vilket var den högsta nivån. Smörsyra och ättiksyra var de dominerande produkterna i det initiala sura pH-värdet 5. Alla tre substraten i batch-reaktorer visade högt produktionsutbyte av ättiksyra och smörsyra. Vid lågt initialt pH var propionsyra den vanligaste syratypen i olja. / Innovative technology, products, and processes are required because of the market's rising need for bioproducts with low carbon footprints. The concept of producing volatile fatty acids (VFAs) via biological processes, primarily through anaerobic mixed culture fermentation, has been revived because of the extensive exploitation of oil resources. The goal of this study was to evaluate the quantity and efficiency of VFA production from simple substrates. The generation of VFA was examined using a batch type acid fermentation system for three simple substrates (oil, starch, and gelatin). We showed that an initial acidic pH was advantageous for acidogenesis and the total generation of VFAs for all three substrates. The VFA production efficiency was achieved and underwent evolutionary development with mixed culture fermentation (MCF)  tests running at an initial acidic pH of 5.0. 7933.08 mgCOD/L butyric acid were produced from starch at pH 3.87 at day 15, which was the highest level. Butyric and acetic acids were the predominant products in the initial acidic pH 5. All the three substrates in reactors showed high VFA production yield of acetic acid and butyric acid. At low initial pH, propionic acid was the most prevalent acid type in oil.

Cellular growth, development, differentiation, and death are mediated to some degree by the interaction of soluble factors with plasma membrane receptors. Traditionally the cellular response to chemical cues has been studied by exposing entire culture dishes to a desired reagent. While the addition of soluble reagents homogenously to cell culture dishes provides a basis for understanding much of cell biology, greater spatial resolution of reagent delivery is necessary in order to elucidate mechanisms on the subcellular scale. This dissertation explores techniques that may improve the quality and precision of delivering soluble factors to cultured cells in order to better understand the complex processes of cell biology. These advancements were made possible by applying high intensity, focused laser light to soluble materials to achieve microscopic three-dimensional (µ-3D) printing. In combination with a previously developed microfluidic cell dosing platform, microstructures were designed and µ-3D printed to hydrodynamically focus reagent streams for cell dosing. Structures were also µ-3D printed within micrometers of living cells from a solution of gelatin and bovine serum albumin with minimal cytotoxicity. When µ-3D printed, these proteins displayed both temperature and pH-responsive properties. In order to allow for on-the-fly control of reagent stream size and temporal pulse width, microstructures were µ-3D printed from temperature-responsive N- isoproplyacrylamide. To further improve the temporal resolution of the system, a technique for cycling between reagents with millisecond exchange times using laminar flow microfluidics was developed. The utility of these techniques was demonstrated by staining rat Schwann cells and mouse neuroblastoma rat glioma hybrid cells (NG108-15) with focused streams of fluorescent dyes. These advancements may allow future experiments to determine the placement of soluble factors necessary for bacterial quorum sensing or stem cell differentiation. / text