Separation and isolation of a desired analyte from an impure sample solution containing numerous unwanted interfering agents is the first step of nearly every laboratory test performed in medicine and biology. Nucleic acids are often of particular interest to doctors and researchers, and although methods currently exist for their isolation, these procedures are costly in time, man-power, and real-estate. In addition to easing the execution of presently performed tests, mitigation or elimination of these drawbacks would make a large range of currently unperformed tests both practical and feasible. This thesis presents a microfluidics-based approach to the isolation of nucleic acids using transverse isotachophoresis (ITP). A boro-silicate glass chip is used with Poly(Acrylamide) gel electrodes to establish an electric field perpendicular to the direction of sample flow, causing a controlled migration of charged particles. The design and fabrication of the microfluidic chip are addressed, along with the development of a transverse-ITP model which predicts the necessary conditions for the successful separation/concentration of an arbitrary sample. Several proof-of-concept images are provided which demonstrate the effectiveness of transverse ITP using surrogate sample inputs. This thesis proposes a direction for future work which aims toward confirming the model presented and preparing the transverse ITP chip to receive biological samples.

Genetic variation at twelve loci which code for enzymes and non-enzymatic protein has been studied by means of starch gel electrophoresis in two apparent species of the pelecypod genus Transennella, Variable allozymes include esterase, leucine aminopeptidase, malate dehydrogenase, phosphoglucose isomerase, tetrazolium oxidase, and nonenzymatic protein, Observed allele frequencies for these systems are in close agreement with Hardy-Weinberg expectations indicating that these protandric hermaphrodites are outbreeding. The genus is characterized by two distinct morphotypes which differ in shell shape and pigmentation. Genetic similarity (I) and genetic distance (D) statistics were computed for the two morphotypes and for two populations of each morphotype. The I and D values derived from intraspecific comparisons of Transennella tantilla populations are 0.99 and 0,004; for Transennella sp., the I value is also 0.99 and D is 0,002. The I and D values obtained from interspecific comparisons (T. tantilla versus T, sp.) are 0,521 and 0,650 respectively. An extensive survey of the phosphoglucose isomerase system was conducted to evaluate microgeographic variation and age-dependent frequency selection at this locus. Differences in allele frequencies were found to be non-significant, suggesting that this locus is without selective import, or that selection for a particular genotype is too slight to be detected with the sample sizes employed, The amount of genetic divergence found between the morphotypes is comparable to interspecific estimates made for a variety of invertebrate species. The results indicate that the morphological divergence apparent in the two morphotypes has been accompanied by substantial genetic differentiation.

In an previous 72-hr mercury bioassay with the larval caddisfly Nectopsyche albida, electrophoretically detectable esterase activity was absent in exposed individuals that succumbed to mercury toxicity, while nine other enzymes remained active hours after death. Esterase activity also persisted in unexposed individuals (Benton and Guttman, 1992a.b). To test the effects of mercury exposure duration on esterase activity, additional larval N. albida were exposed under conditions identical to those in the earlier bioassay, and esterase activity in live individuals was tested electrophoretically every 12 hr. To test the effects of mercury concentration on esterase activity, unexposed N. albida larvae were electrophoresed, and the esterase-specific stain was spiked with various concentrations of mercury. Electrophoretic banding patterns were then densitometrically quantified to identity changes in esterase activity with exposure duration and mercury concentration. Results suggest that: inorganic mercury inhibited esterase activity in N. albida, inhibition increased with exposure duration, and inhibition increased with mercury concentration.

No description available.

博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23513号 / 工博第4925号 / 新制||工||1769(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

In sperm chromatin, DNA exists in a highly condensed state reaching a final volume roughly twenty times that of a somatic nucleus. For the vast majority (>90%) of sperm DNA in mammals, somatic-like histones are first replaced by transition proteins which in turn are replaced by arginine-rich protamines. This near crystalline organization of the DNA in mature sperm is thought crucial for both the transport and protection of genetic information since all DNA repair mechanisms are shut down. Recent studies show that increased DNA damage is linked to dysfunctions in replacing histones with protamines resulting in mispackaged DNA. This increased DNA damage correlates not only to infertility but also impacts normal embryonic development. This damage is currently poorly characterized, but is known to involve oxidative base damage by reactive oxygen species (ROS). Using a variety of biophysical methods, the effect of DNA condensation by polycations on the on free radical access and DNA damage in the packaged state was investigated. In Chapter 2, gel electrophoresis was used to quantify the ability of free radicals to damage both unpackaged and packaged DNA. DNA condensed by polycations shows significantly reduced levels of indirect damage from exposure to free radicals. Combining previous work on packaging density, it is also shown that differences in the packaged state, even by a few Angstroms, can result in significantly different degrees of damage to the DNA. In Chapter 3, we investigate the effects of protamine concentration on the ability to condense and protect DNA. Insufficient protamination is known to be a potential source of protamine dysfunction in mammalian sperm chromatin. Using gel retardation assays and UV-Vis studies, we examined the ability for DNA to condense with protamine at varying nitrogen to phosphate (N:P) charge ratios. Initial results on damage as a function of N:P are also discussed. Future work will more quantitatively determine the interrelationship between DNA packaging densities and the resulting accessibility of DNA to reactive oxygen species (ROS).

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Electrophoresis of solutes was studied in aqueous two-phase systems, concentrating on the special behavior in the interfacial region. Moving boundary electrophoresis was examined in a free fluid U-tube apparatus. Zone electrophoresis was investigated in two-phase systems which were gelled by the addition of acrylamide, which was subsequently polymerized. The size and nature (concentration or dilution) of polarizations which were found to occur was found to depend on the magnitude of the equilibrium partition coefficient of the solute in the two-phase system, as well as the direction of migration across the interface. These polarizations are in addition to those commonly known to occur near regions where electrophoretic flux changes radically, such as near interfaces. They can be a direct result of the requirement for equilibrium across the interface, as demonstrated by our experiments. Models were constructed to numerically simulate this behavior, which accounted for unsteady state electrophoresis and diffusion of multiple proteins or other amphoteric solutes. Two cases were explored, one requiring instantaneous solute equilibration across the interface, the other allowing for a resistance to mass transfer here. All models demonstrated a characteristic noted in experimental studies, concentration at interfaces when electrophoresis is from equilibrium preferred phase towards non-preferred phase. Furthermore, the equilibrium model correctly predicted the complex relationship between partition coefficient, direction of migration, and moving boundary or zone electrophoresis, which causes differences in the polarizations observed in these various systems. The simulation could also quantitatively estimate the width of the polarized region to within an order of magnitude, in comparison with experimental results, while hampered by a lack of mobility data for solutes in solutions containing polymers.