Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.

This study had two objectives: to achieve suspension cultivation of Sf1Ep cells and to develop procedures for achieving the replication of T. pallidum in those cell cultures. Sf1Ep cells have been the sole cell line used for the in vitro cultivation of T. pallidum. A study was undertaken to determine if other cell lines can support growth of T. pallidum. Rabbit skin fibroblasts (RAB-9), nude mouse ear (NME) cells, and normal rebbit testis fibroblasts (RT) were compared to Sf1Ep cells for their ability to support in vitro multiplication of T. pallidum. RAB-9 cells supported multiplication of treponemes equal to that of Sf1Ep cells. NME and RT cells also supported growth but to a lesser extent than Sf1Ep cells. Utilization of alternative cell lines may lead to improved in vitro growth of T. pallidum including possible serial passage.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute and chronic activation of both ionotropic and metabotropic glutamate (glut) receptors is implicated in many neurodegenerative disorders including AD, dementia, epilepsy, stroke and neurotrauma. TRH and glut receptors (ionotropic & metabotropic) receptors are differentially coexpressed in granule and pyramidal neurons of the hippocampus. The author shows TRH to be protective when added to cultured pituitary adenoma (GH-3) cells and neuron-like pheochromocytoma (PC12) cells either prior to, during, or after glut-induced toxicity (Endo. Soc. Abs. 01), and also shows that the possible neuroprotective mechanism may involve heterologous downregulation of the metabotropic glut receptors, using superfused hippocampal slices and noting a reduction of Gαq/11 (SFN Abs. 02). He has also demonstrated that TRH protected against glut toxicity in fetal cortical cultures (Endo. Soc. Abs. 04). To extend these studies he used 14-day cultured rat fetal hippocampal neurons (Day E17) to determine if TRH is protective against toxicity induced by specific ionotropic and metabotropic glut agonists. Neuronal viability and integrity were assessed by trypan blue exclusion and LDH release after 18 hrs following 30 min exposure to glut agonists. Ten µM dihydroxyphenylglycine (DHPG, a Group 1 receptor agonist) + 30 µM N-methyl-D-aspartate (NMDA)-induced toxicity (42% vs contr. P<0.05); whereas, concurrent and continued treatment with 10 uM but not 1uM 3Me-HTRH resulted in less neuronal death and damage (86% vs contr P<0.05; 53% vs contr. P>0.05) respectively. DHPG treatment alone (10 µM) for 30 min. was non-toxic by both criteria (90% vs contr. P<0.05). The data suggest that TRH may be a selective modulator of glut-induced toxicity.

Plant cells resistant to specific amino acid analogs have been reported to accumulate the corresponding free amino acids. The purpose of this study was to determine the concentrations of fifteen free amino acids: alanine, valine, leucine, isoleucine, glutamate, proline, arginine, aspartate, threonine, methionine, lysine, serine, glycine, tryptophan and phenylalanine in Daucus carota cell lines, resistant either to the proline analog azetidine-2 carboxylic acid (A2C), or to the tryptophan analog 5-methyltryptophan (5-MT), or to both the analogs combined. This study also intended to determine if these analogs influence the biosynthesis of the above-mentioned fifteen amino acids in the cell line resistant to A2C and 5-MT. Carrot cell lines resistant to 5-MT, to A2C, or to both the analogs were selected by incubating carrot cells in liquid growth media containing either 0.3 mM 5-MT, or 0.5 mM A2C for 6 to 16 weeks. Free amino acid concentrations were then determined in the extracts of the cells. Resistance to 5-MT resulted in significant increases in the intracellular concentrations of tryptophan, phenylalanine, leucine, valine, isoleucine, and proline. Resistance to A2C resulted in significant increase in proline only. Resistance to both the analogs caused increases in proline, lysine, phenylalanine, and tryptophan concentrations. In the cell line resistant to both the analogs, the treatment with 5-MT caused increases in leucine, proline, aspartate, threonine, lysine, and tryptophan. The treatment with A2C caused increases in isoleucine, arginine, threonine, methionine, lysine, and glycine, whereas treatment with both the analogs caused increases in threonine, lysine, phenylalanine, and tryptophan. These results indicate the possibility of a common biosynthetic control of a number of amino acids in carrot cells, resembling that found in microorganisms. It is also evident from the results that the analogs play an active role in the biosynthesis of amino acids in the resistant cell lines.

No description available.

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Mitochondrial DNA encodes thirteen protein subunits in the oxidative phosphorylation system (OXPHOS) that is responsible for cellular energy production. Mitochondrial disorders have been identified to be associated with mtDNA mutations. However, the molecular mechanisms of specific mtDNA mutations are still being explored in order to establish causative links. This study tries to elucidate the mutational effects of mtDNA on OXPHOS complex activities and cell growths. Using mouse 3T3 fibroblasts as a cell model, single-cell clones with different growth rates were isolated. The entire mtDNA genome was sequenced for mutations. The enzymatic activities of OXPHOS complex I to V were analysed. Three growth patterns represented by five clones were identified. Three clones (clone #2, #3, and #6) had the shortest doubling times (11.5 - 14.9 hours). Clone #1 had a medium growth rate (19.2 hous); and clone #5 had a significantly slow growth rate (22 hours). MtDNA sequencing results revealed that clone #5 had several heteroplasmic mutations (one in 16S rRNA, two in tRNAser (UCN), three in tRNAasp, one in tRNAlys, one in COI, five in COII, and one in ATPase8) while the other four clones showed sequence homology. Enzymatic analyses showed that on average clone #5 had significantly low complex III, IV, and V activities (p < 0.05). Changes in biochemical properties and protein structure were analyzed to deduct possible mechanisms for reduced respiration. In conclusion, the slow growth rate is associated with reduced OXPHOS enzyme functions. It is most likely that the combination of COI and COII mutations resulted in the reduction of complex IV function. It is still unclear whether the ATPase8 mutation (T7869A) in the non-conserved region alone can have such a pronounced phenotypic effect. A reduction in complex III also cannot be explained since there were no mutations in the only mtDNA-encoded complex III gene, but it is possible that there are mutations in the nDNA-encoded complex III genes. Mutations in tRNA and rRNA genes may also be responsible for reduced protein syntheses and consequently reduced OXPHOS activities. It is unclear why complex I activity was not affected. Although the mutational effect of individual mtDNA mutation observed cannot be clearly identified, this study establishes a correlation between mtDNA mutation and cell energy production and growth.