Resonance Raman spectroscopy can be used to estimate the hemoglobin oxygen saturation. Previous studies have demonstrated that exposure of oxygenated red blood cells to laser illumination may result in photo-induced displacement of oxygen from hemoglobin. In this study, an in vitro model was used to evaluate the relationship between this photo-induced effect and the red blood cell velocity. A computer-controlled system was implemented to acquire variables such as red cell velocity, microvessel diameter and hemoglobin oxygen saturation at multiple sites in selected microvascular networks. Using this system, resonance Raman spectroscopy was employed to measure hemoglobin oxygen saturation gradients in arterioles and venules of the rat mesentery in vivo. In several in vitro experimental conditions, there was a significant decrease in photodamage as red cell velocity increased. As blood flowed downstream in arterioles and venules, increased red blood cell velocity was associated with smaller hemoglobin oxygen saturation gradients in vivo.

Sickle cell anemia is caused by a single mutation in the β-hemoglobin gene, HBB. The disease originated in Africa and affects millions of people worldwide. Sickle hemoglobin tetramers polymerize upon deoxygenation and lead to hemolysis and vaso-occlusion. Patients with high fetal hemoglobin (HbF) can have milder disease. The only FDA-approved drug is hydroxyurea that increases HbF. HbF modulates the disease by preventing the polymerization of sickle hemoglobin and reduces the pain episodes, anemia, and organ damage associated with the disease. There are five common haplotypes associated with the HbS gene and that are very loosely associated with disease severity and HbF. Understanding the genetic bases of HbF regulation is a key factor to identify potential drug targets to induce HbF for therapeutic purposes. To fully understand the mechanism behind HbF regulation, developing a fast and accurate computational method for sickle cell haplotype classification is useful for examining the variability of HbF among sickle cell patients. Moreover, investigating the cis and trans-acting regulators of HbF gene expression to pinpoint the mechanism through which they regulate HbF is essential to develop a successful treatment. The availability of high-throughput genetic data provides an excellent opportunity to study HbF regulation in sickle cell patients and normal people comprehensively. The work reported in this thesis describes a fast and accurate method for sickle cell HBB haplotype classification. I also examine the differential effect of cis and trans-acting HbF hemoglobin regulators on -globin gene expression using the GTEx database and identify BCL2L1 as a new potential trans-regulator of HbF.

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Background: Knowledge regarding the associations of (i) hemoglobin level (Hb) prior to cancer diagnosis and cancer mortality (ii) the full range of Hb and cancer incidence and (iii) baseline inflammatory/other biomarkers and Hb in older populations is limited. The present study examined the associations of anemia status/Hb with cancer incidence and mortality, as well as the association with inflammatory biomarker levels in post-menopausal women. Methods: Anemia was defined as Hb <1 2 g/dl, while high Hb was defined as Hb >= 15 g/dl, or >= 16 g/dl. Associations were determined in three Women's Health Initiative Study sub-populations. The association between anemia/Hb with cancer mortality was determined in women without (N=21,021) or with (N=2,976) cancer history who had cancers on follow-up. The cross-sectional association of biomarkers and anemia/Hb was determined on 1,001 women with these available data. Finally, the association between anemia/Hb with cancer incidence was determined in women enrolled in the Observational Study/Clinical Trial who did not have a history of cancer/extreme energy intakes/missing follow-up time (N=140,269). Results: Anemia was associated with a 21% higher hazard of total cancer death in participants with, and a 55% greater hazard in participants without cancer history. Anemic women with a history of cancer had twice the hazard of colorectal cancer death. C-reactive protein, TNF-alpha, TNF-beta and TNFR2 were significantly associated with anemia. IL-1 alpha and IL-10 were significantly associated with continuous Hb. Anemia was not associated with cancer incidence in the total population, but anemic African-American women had a reduced risk of any cancer incidence which was not observed in white women (p-interaction=0.03). Women with high Hb had an increased hazard of any (HR: 1.37; 95% CI: 1.17, 1.60) or breast cancer (HR: 1.42; 95% CI: 1.10, 1.84) incidence. Conclusions: Anemia determined prior to cancer diagnosis was associated with total and colorectal cancer death. High Hb was associated with increased risk of total cancer and breast cancer incidence. Anemia was associated with elevated levels of C-reactive protein, TNF-alpha, TNF-beta and TNFR2, while continuous Hb was associated with IL-1 alpha and IL-10. Further research is required to confirm associations and clarify causal mechanisms.

Hemoglobin, the oxygen-carrying component of red blood cells, is known as a nitric oxide (NO) chelating agent. For this reason, hemoglobin has been used widely in studying the role of nitric oxide in long-term potentiation (LTP) and excitotoxicity. However, the direct electrophysiological actions of hemoglobin has not been examined. In this investigation, the actions of hemoglobin on rat hippocampal CAl neurons were studied since hemoglobin may be present in hemorrhagic stroke and other head injuries. Superfusion of rat hippocampal slices with 0.1 mM of bovine hemoglobin for 15 minutes was induced a significant depolarization associated with an increase in the input resistance. In addition, hemoglobin suppressed the evoked synaptic responses and increased the depolarization-induced discharge of action potentials, of rat hippocampal CAl neurons. These hemoglobin-mediated changes usually recovered partially 30 minutes after the removal of hemoglobin. While the depolarizing action of hemoglobin was enhanced in a calcium-free medium, it was not significantly changed by 2-amino-5-phosphonovalerate (APV) and 6- cyano-7-nitroquinoxaline-2,3-dione (CNQX). These observations suggest that the depolarizing action of hemoglobin is independent of the presence of extracellular calcium and activations of the excitatory amino acid receptors. Because hemoglobin has been observed to suppress the depolarizing action of glutamate, it is possible that hemoglobin suppresses the EPSP by interfering with the actions of glutamate. Although hemoglobin has been suggested to suppress LTP and excitability by scavenging nitric oxide (Garthwaite et al., 1988; Haley et al., 1992; 0’ Dell et al., 1991; Schuman and Madison, 1991), the reported actions of hemoglobin were not removed by pre-treatment with 100 pM or 500 pM of No-nitro-L-arginine, a nitric oxide synthase inhibitor. Similar to the scavenging property of hemoglobin, the iron content of hemoglobin probably did not contribute to the actions of hemoglobin since 0.4 mM or 2.0 mM of ferric chloride did not simulate the effects of hemoglobin. Because neurons can be exposed to hemoglobin in hemorrhagic stroke and head injuries, the electrophysiological actions of hemoglobin on rat hippocampal CAl neurons may be relevant to the neurological complications associated with intracranial hemorrhage and head injuries. Further studies on mechanisms of the electrophysiological actions of hemoglobin are necessary for understanding the role of hemoglobin in neuronal damages associated with hemorrhagic stroke and other head injuries.

In recent years there has been considerable interest in the development of microwave-based food processing technologies. Microwave radiation is considered to have both thermal and non-thermal effects. The thermal effects are related to the heat generated by the absorption of microwave energy by water or by organic molecules, but very little is known about the mechanisms involved in the putative non-thermal effects. It has been postulated that the latter could involve a direct energy transfer from the electromagnetic field to the vibrational modes of macromolecules, altering their conformation. In the present study, the non-thermal effects induced upon irradiation of protein solutions with microwave energy were investigated by employing Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy to elucidate the unfolding pathways of hemoglobin and beta-lactoglobulin (5% in D2O) subjected to either microwave irradiation (2450 MHz) or conventional heating. (Abstract shortened by UMI.)

In early rainbow trout development, a switch from high-affinity embryonic hemoglobin to lower-affinity adult hemoglobin occurs along with a turnover of round, embryonic erythrocytes to oval, adult erythrocytes. The objective of my thesis was to determine how the ontogeny of blood oxygen transport was affected by chronic hypoxia (30% of saturation) in rainbow trout. Hemoglobin-oxygen affinity, cooperativity, and the Bohr and Root effects were unaffected by hypoxia treatments, whereas hemoglobin content, erythrocyte counts, and hematocrit were significantly reduced. In hypoxia, larvae had higher concentrations of embryonic hemoglobin mRNA and embryonic erythrocytes than stage-matched normoxia-reared larvae. Overall, these results indicate that chronic hypoxia suppresses erythrocyte development prior to complete yolk absorption. Ultimately, this suggests that in early ontogeny rainbow trout conform to low oxygen conditions, rather than mounting the hypoxia response observed in oxygen-regulating adult trout.

Polymorphisms of platelet glycoproteins (GPs) are frequently targets for anti-platelet antibodies. At least 19 antigenic polymorphisms have been identified on platelet GPs. Antibodies against the HPA-lb polymorphism (a Leu to Pro switch at amino acid residue 33 of the IIIa sub-unit of GP IIb/Illa) have been attributed to as much as 90% of all cases of neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura in caucasians. The HPA-lb polymorphism has also been equivocally associated with coronary artery disease, particularly early onset (<60 years of age) myocardial infarction. Current technology for identifying individuals with the HPA-lb phenotype is limited to the labor-intensive, highly technical and expensive process of DNA amplification by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) analysis.This study proposes an alternative method for phenotyping individuals for the HPA-1 polymorphism using the Biocytex Platelet HPA-1 kit. The kit identifies the HPA-1 polymorphism utilizing two monoclonal CD61 (platelet glycoprotein IIb/IIIa) antibodies, one of which has a lowered affinity for GP llb/Illa possessing the HPA-lb polymorphism. Fluorescent labeling of bound antibody allows for flow cytometric quantitation of antibody binding capacity (ABC) for both monoclonal antibodies, and ratios derived from the ABC can be used to phenotype previously unknown samples.The Biocytex HPA-1 kit identified 73 of 74 (98.6%) individuals possessing the HPA1 a/HPA-1 a phenotype, 22 of 22 (100%) HPA-1 a/HPA-1 b individuals and 4 of 4 (100%) HPAIb/HPA-Ib individuals. All HPA-lb phenotypes were confirmed by PCR/RFLP. Total accuracy of the test system was 99%. / Department of Biology