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xv, 178 leaves : ill. ; 28 cm Typescript. (Another copy on microfilm is located in Archives) Thesis (Ph.D.) -- University of Oregon Includes vita and abstract Bibliography: leaves 163-178 University of Oregon theses, Dept. of Biology, Ph.D., 1980

Diabetic individuals have been found to have consistently higher levels of a minor hemoglobin component, HbAIc, than non-diabetic individuals. Previous investigators have suggested that variation in these high levels of HbAIc may be a reflection of the degree of diabetic hyperglycemia, of hypertriglyceridemia in diabetes, and of diabetic control. To date, evaluation of HbAIc as a clinically useful parameter has been hampered by the complexity of the method of measurement, the inconsistency in ranges of normal values reported, and the lack of a broad data base for comparison with new results. This investigation began with a critical appraisal of the methods currently in use for measurement of HbAIc, followed by a simplification and standardization of theJ assay. Then the levels of HbAIc in 16 non-diabetics and 47 diabetics were determined and the mean values for these 2 groups compared. The relationships between HbAIc levels in the diabetics and selected clinical data |fasting blood sugar, 24-hour urinary sugar, age,duration of illness, dietary record, insulin dosage, and family history of diabetes) were examined. Finally, the degree of diabetic control in each of the diabetic patients was estimated by the attending physician on a scale of 1 to 5 (1=very good, 2=good, 3=fair, 4=poor, 5=very poor) and was compared with the HbAIc measurement. Results of the investigation have shown that the chromatographic measurement of HbAIc is unusually sensitive to the pH of developers used and also somewhat variable with respect to the length of storage time and the optical density at which samples are read. The comparison of mean values of HbAIc for diabetics and non-diabetics has confirmed the approximately twofold higher concentration of HbAIc in diabetics. Significant correlations were found between HbAIc and fasting blood sugar (r = .442), fat content of diet (r=-. 300) , family history of diabetes (r=-.312) and degree of diabetic control (r=.529). Thus, HbAIc values tend to be higher in patients whose fasting blood sugar is high, whose diet contains relatively little fat, whose relatives are diabetic and /or whose diabetic control is poor. Correlations between HbAIc and duration of diabetes, HbAIc and insulin dosage or HbAIc and 24-hour urinary sugar were not statistically significant (r=-.131, r=-.264, r=„067, respectively). The HbAIc level appears to be an accurate reflection of fasting blood sugar levels averaged over a prolonged period of time (r=.587). In conclusion, HbAIc levels were found to provide an objective measure of diabetic control. The improved assay method makes it a practical and valuable tool for the clinician as well as the investigator. Measurement of HbAIc levels in diabetics presents a considerable advantage over currently available measures of diabetic control in assessing the long-term effectiveness of diabetic management. / Land and Food Systems, Faculty of / Graduate

This work focuses on pigmy sperm whale and horse myoglobins (Mbs), which are distinguished by a single heme pocket residue variant in the CD3 position, when the heme iron is in the +3 oxidation state (i.e. the met form). The strategy employed is as follows: (i) assign heme peripheral protons; (ii) assign the amino acid residues from the heme cavity; (iii) assess the dynamics of ligand binding in the active site by means of hydrogen Iability, solvent isotope effects, and heme-insertion isomer trapping, all by NMR methods. The results of these studies portray dynamic solution structure of the Mb ligand binding site, and provide a set of standard parameters for the studies of larger hemoproteins. The findings are also important for understanding protein-ligand interactions in general. My research investigates the mixed spin metazido and metimidazole complexes of Mbs for the following reasons. First, the allosteric properties of hemoglobin arise mainly from the transition between its two possible quaternary structures. This can be studied by paramagnetic NMR because it is one of the most sensitive tools in terms of changes in the molecular and/or electronic structure of the heme. Second, both the N₃- and imidazole (lm-) complexes are good compromises, in terms of sizes, between the small diatomic oxygen or CN⁻ molecules and the bulky phenyl group. Thus, we can determine the influence of ligand size on structural perturbation of the Heme crevice by comparison among the different size groups. Third, the saturation-transfer phenomenon between metMbIm and metMbH₂0 provides a route to assignments in metMbH₂0 by using assignments of metMbIm. This is crucial because metMbH₂0 is the basis of theoretical calculations of the isotropic shift due to axial ligand field in pure high-spin hemoproteins. Finally, the importance of the metMbIm is underscored by the fact that it is a bis-imidazolium complex, which can then serve as a model other bis-histidyl proteins. Most of the heme peripheral resonances of metEqMbIm and metEqMbN₃ were identified by means of two-dimensional NOESY,COSY, and EXSY spectroscopy. The strongly relaxed upfield protons in metMbIm were assigned based on steady-state 1D NOE and T₁ experiments. Based on the results from metMblm in which saturation transfer of one upfield resonance led to two different free ligand peaks, bound Im equilibration was envisioned and proven by the divergence of broad downfield heme methyl peaks into two peaks each, showing distinctive population preference of each isomer. Dicyanoheme probe, as well as hydrogen Iability comparison studies between pigmy sperm whale Mb and horse Mb in the azido and imidazole states, asserts that single variant pocket residue CD3 is crucial in gating the ligand mobility into and out of the active site. The assignments of heme peripheral and upfield resonances enabled the subsequent assignments of some heme pocket amino acid residues. The facile exchange of bound Im with solvent H₂0 lays the ground work for identification of heme pocket residues in metMbH₂0. Furthermore, while deuterated heme previously allowed only assignment of the non-diastereomeric specific heme 2-vinyl β proton, saturation-transfer from horse imidazole Mb affords the specific identification of 2Hᵦt.

No description available.

Thesis / Master of Science (MS)

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.

Call number: LD2668 .T4 1930 C64

BACKGROUND:The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.RESULTS:We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.CONCLUSIONS:These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

The order Sirenia is one of only two mammalian groups to have completely forgone terrestrial life. While extant sirenians are confined to the tropical waters, the recently extinct Steller’s sea cow (Hydrodamalis gigas) evolved to exploit the frigid waters of the North Pacific Ocean. As limits on O2 availability during submergence and decreased tissue temperature impose severe constraints on oxygen delivery, the oxygen binding (globin) proteins of sirenians are expected to have evolved under strong selection pressures. My comparative molecular analyses indicate that selection pressures on two globin genes (HBA and HBZ-T1) increased in transitional sirenians. As these genes encode the α-chains of all Hb isoforms throughout sirenian development, the resulting functional consequences to adult sirenian Hbs were tested using recombinant Hbs of Steller’s sea cow, the dugong (Dugong dugon), their last common ancestor, and the Florida manatee (Trichechis manatus latirostris). These tests reveal that high affinity Hbs—exceeding those of other mammals examined to date—arose early in sirenian evolution, presumably to maximize O2 extraction from the lungs and limit premature O2 offloading during submergence. Moreover, I demonstrate that the Hb–O2 affinity of the extinct sub-Arctic Steller’s sea cow is less affected by temperature than other sirenians, safeguarding O2 delivery to cool peripheral tissues. However, while this phenotype has primarily been attributed to the binding of additional allosteric effectors to the Hb moiety, Steller’s sea cow Hb binds relatively few of these ligands. Instead, my results suggest the thermodynamic properties of discrete allosteric effector sites are altered by epistatic interactions, a phenomenon that appears to be a critical component to cold adaptation in mammalian Hbs. As the HBA and HBZ-T1 loci also encode sirenian prenatal Hbs, the functional properties these proteins were tested to reveal their O2 affinity increased in parallel to maternal Hb. Notably, Steller’s sea cow HbF has the highest reported O2 affinity of any mammalian Hb tested to date. As the HBA gene encodes the -subunit of both the prenatally expressed HbF and adult expressed HbA proteins, the molecular remodeling of this locus may have concurrently increased the affinity of each protein. / February 2017