Protein-ligand and protein-protein interactions are critical to cellular function. Most cellular metabolic and signal tranduction pathways are influenced by these interactions, consequently molecular level understanding of these associations is an important area of biochemical research. We have examined the thermodynamics of several protein-protein associations and the protein-ligand interactions that mediate them. Using Fluorescence Correlation Spectroscopy, we have examined the putative interaction between pig heart malate dehydrogenase (MDH) and citrate synthase (CTS). We demonstrate a specific, low-affinity interaction between these enzymes. The association is highly polyethylene glycol (PEG)-dependent, and at high concentrations of NaCl or PEG, non-specific aggregates are formed. We demonstrate that oxaloacetate, the intermediate common to both CTS and MDH, induces the association at concentrations below the Km of CTS, suggesting that the open conformation of CTS is involved in the association. Using several biophysical techniques, we have examined the subunit associations of B. stearothermophilus phosphofructokinase (PFK). We demonstrate that the inhibitor bound conformation of the enzyme has reduced subunit affinity. The kinetics and thermodynamics of the phosphoenolpyrvuate (PEP)-induced dissociation of PFK have been quantified. Binding substrate, fructose-6-phosphate (F6P), stabilizes the enzyme to inhibitor-induced dissociation by 132-fold. These data suggest that subunit associations may play a role in the allosteric inhibition of PFK by PEP. The thermodynamics of the protein-ligand associations and allosteric inhibition of E. coli phosphofructokinase have been examined using intrinsic fluorescence and hydrostatic pressure. Both ligand-binding affinity and PEP inhibition are diminished by pressure, whereas substrate-binding affinity for inhibitor-bound enzyme is pressure-insensitive. Larger entropic than enthalpic changes with pressure lead to the overall reduction in free energies. Using a fluorescence-based assay, we have developed a series of baroresistant buffer mixtures. By combining a buffer with acid dissociation of negative volume with a buffer of positive volume, a pressure-resistant mixture is produced. Alteration of the molar ratio of the two component buffers yields mixtures that are pressure-insensitive at pH values around neutrality.

Several research groups have applied elevated hydrostatic pressure to ONH astrocytes cultured on a rigid substrate as an in vitro model for glaucoma. These studies have shown significant biological effects and this hydrostatic pressure model is now becoming generally accepted in the ophthalmic community. However, since the applied pressures were modest the finding of significant biological effects due to pressure alone is surprising. We hypothesized that the application of hydrostatic pressure as described in these studies also altered gas tensions in the culture media. Our goal was to design equipment and carry out experiments to separate the biologic effects of pressure from those of hypoxia on cultured astrocytes. We designed equipment and carried out experiments to subject cultures of DITNC1 astrocytes to the four combinations of two levels of each parameter. We explored the morphology and migration rates of astrocytes, but observed no significant change in any of these properties.

The growing demand for simulated fish products coupled with the declining stocks of traditional fish species for making these products, have increased efforts aimed at developing novel procedures to put non-traditional fish species like bluefish (Pomatomus saltatrix) and tilapia (Tilapia nilotica) to better economic use. Two procedures which fulfill both objectives are (i) application of high hydrostatic pressure, and (ii) utilization of protease inhibitor to control fresh fish spoilage due to microbial activity and/or autolysis by endogenous enzymes. High hydrostatic pressure has been used to formulate gels with superior functional properties, while $ alpha sb2$-macroglobulin, a broad spectrum protease inhibitor, has been added to control proteolysis in fish gels during processing and storage. / In this study, high pressure was applied at levels of 300 to 3,742 atm for 30 min to formulate gels from bluefish meat paste, and the properties of the resulting gels were compared with those of heat-induced gels formulated at 90$ sp circ$C for 20 min or 60$ sp circ$C for 60 min. / The effects of $ alpha sb2$-macroglobulin and cooking temperatures on the properties of tilapia gels were also studied. (Abstract shortened by UMI.)

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Dallas G. Hoover, Dept. of Animal & Food Sciences. Includes bibliographical references.

Thesis (Ph. D.)--Ohio University, June, 2000. / Title from PDF t.p.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 169-175). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 169-175). Also available on the Internet.

Thesis (M.S.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 128-132).

Orientador: Carlos Francisco Sampaio Bonafé / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T11:09:06Z (GMT). No. of bitstreams: 1 Norberto_DouglasRicardo_M.pdf: 4245356 bytes, checksum: 8a2d8041749dec81554527bd8729d8ea (MD5) Previous issue date: 2007 / Resumo: A hemoglobina extracelular de Glossoscolex paulistus (eritrocruorina) foi investigada com respeito ao efeito da temperatura na dissociação induzida por alta pressão hidrostática. O aumento de temperatura e pressão induziu o processo de dissociação, como observado pela significativa diminuição da intensidade de espalhamento de luz. Tais informações foram confirmadas através de HPLC em gel filtração e microscopia eletrônica. Ocorreu uma redução dos valores de energia livre de Gibbs de dissociação indicando um processo predominantemente endotérmico. A variação de entalpia (?H) observada no processo foi de 27,82 MJ/mol de hemoglobina (Hb) e a de entropia (T?S), à temperatura de 293 K, de 23,91 MJ/mol de Hb. Foi encontrada uma redução da mudança de volume de dissociação (?V) de -78,20 para -10,44 mL/mol de subunidade de Hb. Em condições atmosféricas e temperatura de 293 K, a variação da energia interna de dissociação (?U) foi de 27,82 MJ/mol de Hb e da energia livre de Helmholtz, (?A), de 3,92 MJ/mol de Hb. Os resultados da dissociação oligomérica em alta pressão no intervalo de temperatura investigado mostraram a ocorrência de etapas distintas de mudança de estabilidade conformacional. Complementarmente, foi realizado um estudo das interações intersubunidades e da área de exposiçao da proteína ao solvente no processo de dissociação, permitindo a obtenção de importantes dados quantitativos no processo / Abstract: Glossoscolex paulistus extracelluar hemoglobin (erithrocruorin) was studied with respect of thermal effect on the dissociation induced by high hydrostatic pressure. The increase of temperature and pressure led to dissociation process, as observed by the significant decrease in the intensity of light scattering values. Such information was confirmed by HPLC gel filtration and electron microscopy. A predominantly endothermic process was observed with the reduction in the Gibbs free energy of dissociation. The enthalpy change (?H) obtained was of 27,82 MJ/mol of hemoglobin (Hb) and the entropy change (T?S), at a temperature of 273 K, 23,91 MJ/mol of Hb. The estimated volume change of dissociation (?V) decreased from -78,20 to -10,44 mL/mol of subunit of Hb. The change of internal energy of dissociation (?U), at atmospheric conditions and temperature of 293K, was of 27,82 MJ/ mol of Hb and the change of free energy of Helmholtz (?A) was of 3,92 MJ/mol of Hb. The results also indicated that the dissociation of oligomeric Hb at high pressure and at investigated temperature range occurs in distinct steps of conformational stability and allowed to obtain significant quantitative data in the process. In addition, it was studied the subunity interactions and related exposed area of the protein solvent dissociation, attempting to obtain an quantitative description of the process / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular