Indiana University-Purdue University Indianapolis (IUPUI) / Tissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.

<p> In plants, phosphocholine (PCho) is a precursor to the membrane component phosphatidylcholine (PtdCho) and free choline (Cho). A mutant Saccharomyces cerevisiae yeast strain unable to produce PtdCho without exogenous choline was used for transformation with an Arabidopsis cDNA library cloned in the yeast expression vector pFK61. A plant cDNA associated with locus At1g48600 functionally complemented the mutant by restoring growth on minimal synthetic medium lacking choline but containing the phosphobase phosphomethylethanolamine (PMEA). Crude extracts prepared from the yeast showed a novel capacity to convert PMEA to phosphodimethylethanolamine (PDEA) and PCho and hence this enzyme has been named Arabidopsis S-adenosyl-L-methionine (AdoMet): phosphomethylethanolamine N-methyltransferase (AtPMEAMT). </p> <p> AtPMEAMT is a bipartite enzyme containing tandem N-and C-terminal AdoMet-binding domains. The predicted amino acid sequence shows an 87% identity to the previously characterized AdoMet: phosphoethanolamine N-methyltransferase (AtPEAMT) from Arabidopsis. An important distinction between AtPMEAMT and AtPEAMT is that the former enzyme is unable to methylate phosphoethanolamine (PEA). However, both AtPEAMT and AtPMEAMT can methylate PMEA and PDEA, two phosphobase intermediates ofPCho synthesis. The apparent Km values were determined for AtPEAMT and AtPMEAMT toward PMEA and PDEA and found to be 0.32 and 0.14 mM, respectively, for PEAMT and 0.16 and 0.03 mM, respectively, for PMEAMT. The N-and C-terminal Ado Met-domains of PEAMT and PMEAMT were cloned separately into a pET30a(+) vector for protein expression and extracts containing recombinant proteins were assayed for phosphobase methyltransferase activity. Only the gene product encoding the domain associated with the C-terminal half of PMEAMT methylated both PMEA and PDEA, an activity found with the native protein. A chimera was produced by combining the N-terminal half ofPEAMT and the C-terminal half of PMEAMT. The chimeric protein is able to methylate PEA, PMEA and PDEA indicating that a feature associated with the N-terminal half of PEAMT is required for PEA methylation. This result suggests that differences associated with the N-terminal domain are likely responsible for the inability ofPMEAMT to use PEA as a substrate. </p> <p> An Arabidopsis mutant line with a T-DNA insertion in the promoter region of PMEAMT (SALK 006037) was obtained and RT-PCR analysis of plants homozygous for the insert showed that the mutant lacks transcripts associated with this gene. Relative to wild-type plants grown under identical conditions the mutant plants showed no visible difference in morphological or developmental phenotype. However, shotgun lipidomics using electrospray ionization tandem mass spectrometry showed a 2.1-fold greater abundance ofa 34:3 phosphatidylmethylethanolamine (PtdMEA) molecular species in mutant plants compared to wild-type. One biological role of PMEAMT may be to reduce the likelihood for PtdMEA incorporation into phospholipids ofmembranes. PtdMEA incorporation in membranes is associated with reduced viability of yeast but its effect on the physiology ofplants is, as yet, unknown. </p> / Thesis / Doctor of Philosophy (PhD)

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<p>Investigating the presence of microbial communities in the Earth's deep terrestrial subsurface and the metabolic processes taking place in these environments provides insight into the some of the ultimate limits for life on Earth, as well as the potential for microbial life to exist within the subsurface of other planetary bodies. This Master's thesis project utilized phospholipid fatty acid (PLFA) analysis, in combination with carbon isotope analyses (δ¹³C and Δ¹⁴C), to explore the presence and activity of microbial communities living within deep terrestrial subsurface fracture water systems and low permeability, deep sedimentary rocks. Deep fracture water systems, ranging from 0.9 to 3.2 km below land surface, were sampled for microbial communities via deep mine boreholes in the Witwatersrand Basin of South Africa. PLFA concentrations revealed low biomass microbial communities, ranging from 2x10¹ to 5x10⁴ cells per mL and the PLFA profiles contained indicators for environmental stressors, including high temperatures and nutrient deprivation. δ¹³C and Δ¹⁴C analyses of PLFAs and potential carbon sources (dissolved inorganic carbon (DIC), dissolved organic carbon (DOC) and methane) identified microbial utilization of methane in some systems and utilization of DIC in others. Evidence for microbial oxidation of methane and chemoautotrophy in these systems is consistent with a self-sustaining deep terrestrial subsurface biosphere that is capable of surviving independent of the photosphere. Viable microbial communities were also identified within deep (334 to 694 m depth) sedimentary rock cores sampled from the Michigan Basin, Canada. PLFA analyses revealed microbial cell densities ranging from 1-3 x 10⁵ cells/mL and identified PLFA indicators for environmental stressors. These results demonstrate the ubiquity of microbial life in the deep terrestrial subsurface and provide insight into microbial carbon sources and cycling in deep microbial systems which may persist in isolation over geologic timescales.</p> / Master of Science (MSc)

Two-dimensional (2D) nanoscale confinement effects on poly(ε-caprolactone) (PCL) crystallization were probed through crystallization studies of PCL-b-poly(tert-butyl acrylate) (PCL-b-PtBA) copolymers, PCL with bulky tri-tert-butyl ester endgroups (PCL triesters), PCL with triacid end groups (PCL triacids), and magnetic nanoparticles stabilized by PCL triacid (PCL MNPs) at the air/water (A/W) interface. Thermodynamic analyses of surface pressure-area per monomer (Π−A)) isotherms for the Langmuir films at the A/W interface showed that PCL-b-PtBA copolymers, PCL triheads and PCL MNPs all formed homogenous monolayers below the dynamic collapse pressure of PCL, Π_C ~11 mN•m⁻¹. For compression past the collapse point, the PCL monolayers underwent a phase transition to three-dimensional (3D) crystals and the nanoscale confinements impacted the PCL crystalline morphologies. Studies of PCL-b-PtBA copolymers revealed that the morphologies of the LB-films became smaller and transitioned to dendrites with defects, stripes and finally nano-scale cylindrical features as the block length of PtBA increased. For the case of PCL triester, irregularly shaped crystals formed at the A/W interface and this was attributed to the accumulation of bulky tert-butyl ester groups around the crystal growth fronts. In contrast, regular, nearly round-shaped lamellar crystals were obtained for PCL triacids. These morphological differences between PCL triacids and PCL triesters were molar mass dependent and attributed to differences in dipole density and the submersion of carboxylic acid groups in the subphase. Nonetheless, enhanced uniformity for PCL triacid crystals was not retained once the polymers were tethered to the spherical surface of a PCL MNP. Instead, the PCL MNPs exhibited small irregularly shaped crystals. This nano-scale confinement effect on the surface morphology at the A/W interface was also molar mass dependent. For the small molar mass PCL MNPs, two layers of collapsed nanoparticles were observed. In a later chapter, studies of polyethylene glycol (PEG) surfactant adsorption onto phospholipid bilayers through quartz crystal microbalance with dissipation monitoring (QCM-D) measurements revealed a strong dependence of the adsorption and desorption kinetics on hydrophobic tail group structure. PEG surfactants with a single linear alkyl tail inserted and saturated the bilayer surface quickly and the surfactants had relatively fast desorption rates. In contrast, PEG lipids, including dioleoyl PEG lipids and cholesterol PEGs, demonstrated slower adsorption and desorption kinetics. The interactions of Pluronics and Nonoxynol surfactants with phospholipid bilayers were also studied. Pluronics showed no apparent affinity for the phospholipid bilayer, while the Nonoxynol surfactants damaged the lipid bilayers as PEG chain length decreased. / Ph. D.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第25222号 / 薬科博第184号 / 新制||薬科||21(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 井垣 達吏, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Investigation of life’s limitations on Earth provides the necessary information to constrain where life outside of Earth may be proliferating or previously existed. This Master’s thesis applied phospholipid fatty acid (PLFA) analysis in combination with organic carbon and 16S rRNA gene data to assess and characterize microbial communities through both microcosms and in situ samples of bentonite clay, which is an intended barrier component for the long-term storage of high-grade nuclear waste. Microcosm experiments were set up to test the impact of water activity in as-received, uncompacted bentonite clays using a high (0.99) and low (0.93) water activity over a one month period. Under aerobic incubation water activities of 0.93 and 0.99 had no resolvable effect between water activity levels on the growth of cells of indigenous communities of microbes in as-received uncompacted bentonite. Growth was detected under both water activities by a significant increase in total PLFA abundance. The increase in PLFA over the period of the study suggested an approximate increase in cells from 4x10^6 to 2x10^7 E.coli equivalent cells/g. The distribution of the PLFA and genetics data suggests the community is composed predominantly of gram-positive aerobic heterotrophs with lesser amounts of anaerobic bacteria and eukaryotes potentially in the form of fungi. Similar cell abundances and community structures were identified in the Tsukinuno Mine bentonite DGR analogue site which is a ~12 to 16 Ma deposit approximately 200 m below the surface. Total PLFA recovered from the core subsamples ranged from 32 pmol PLFA/g to 431 pmol PLFA/g, which corresponds to a range from 7.5x10^5 to 1.2x10^7 E.coli equivalent cells/g, across all cores. The community was composed of both aerobic and anaerobic bacteria consisting of gram-positive and gram-negative bacteria, as well as possible sulfate-reducing bacteria and eukaryotes. / Thesis / Master of Science (MSc)