Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2003. / CD-ROM reproduces dissertation in PDF format; Adobe Acrobat required. Includes bibliographical references. Also available on the Internet.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Obesity is closely related to many medical complications such as type 2 diabetes, hypertension and heart failure. Obesity and other factors, including elevated blood glucose levels, hypertension, and dyslipidemia, constitute a constellation of symptoms known as the metabolic syndrome, which are the risk factors for coronary artery disease. Lipocalin-2 is a pro-inflammatory adipokine causally involved in the development of obesity-associated metabolic and cardiovascular diseases. Recent clinical and experimental evidences demonstrate an association between augmented circulating lipocalin-2 and cardiac dysfunction. However, little is known about the detailed roles of lipocalin-2 in regulating pathophysiological functions of the heart. The present study was designed to compare the heart functions of mice with normal (WT) or deficient lipocalin-2 (Lcn2-KO) expression and to examine the molecular mechanisms underlying lipocalin-2-mediated deteriorated effects in hearts. Echocardiographic analysis revealed that the myocardial contractile function was significantly improved in hearts of Lcn2-KO mice, under both standard chow and high fat diet conditions. The heart function before and after I/R injury (20-min of global ischemia followed by 60-min of reperfusion) was assessed using the Langendorff perfusion system. Compared with WT littermates, hearts from Lcn2-KO mice showed improved functional recovery and reduced infarct size following I/R. These phenomena can be observed in mice under both standard chow and high fat feeding conditions. Under baseline condition, the mitochondrial function of hearts from Lcn2-KO mice was significantly enhanced, as demonstrated by biochemical analysis of respiratory chain activity, markers of biogenesis and oxidative stress, as well as electron microscopic investigation of the mitochondrial ultrastructure. Acute or chronic administration of lipocalin-2 impaired cardiac functional recovery to I/R and dampened the mitochondrial function in hearts of Lcn2-KO mice. These effects were associated with an extensive modification of the fatty acyl chain compositions of intracellular phospholipids. In particular, lipocalin-2 facilitated the redistribution of linoleic acid (C18:2) among different types of phospholipid, including cardiolipin, which is exclusively located in the mitochondria inner membrane. The direct effects of lipocalin-2 on both H9c2 and NCM cells were also examined. TUNEL assay and flow cytometry analysis demonstrated that lipocalin-2 treatment promoted apoptosis in cardiomyocytes. Lipocalin-2 induced an early phase of phosphatidylserine exposure, followed by Bax-translocation and caspase-3 cleavage. The results collectively suggested that lipocalin-2 initiated the intrinsic mitochondria-mediated apoptotic pathway. In the hearts of Lcn2-KO mice, significantly reduced number of apoptotic cells was observed after I/R injury. In conclusion, lacking of lipocalin-2 improved heart function recovery during I/R injury via mitochondrial function restoration, phospholipids remodeling, and inhibition of cardiomyocytes apoptosis. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Obesity is one of the leading causes of world-wide life-threatening diseases, such as type 2 diabetes mellitus, atherosclerosis and cardiovascular diseases. So far, there is no effective, promising and safe remedy. Development of cost-effective anti-obesity therapies and promising biomarkers for obesity-related diseases have become a demanding task. Lipocalins, such as LCN13, were recently identified as potential drug target because of its beneficial effects on glucose and fat metabolism in mouse. LCN14 is a putative lipocalin that share high degree of homology with LCN13. In this study, it is experimentally proved that LCN14 is a secretory protein that is mainly expressed in white adipose tissues. It is also demonstrated that serum LCN14 level was significantly increased in mice with HFD treatment, and it was significantly reduced in diet-induced obese and diabetic (db/db) mice. In addition, the degree of suppression of circulating LCN14 was greater than that of LCN13 in diet-induced obese and db/db mice. Therefore, serum LCN14 level could be a promising marker for risk prediction of obesity and its complications. Further investigation is urgently needed to unveil the important roles of LCN14 in metabolism. / published_or_final_version / Medicine / Master / Master of Medical Sciences

abstract / Microbiology / Doctoral / Doctor of Philosophy

An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env). Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development. I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively. 454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans. Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure. In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

alpha- and beta-dystroglycan (DG) were first identified as members of an oligomeric, transmembrane complex expressed in muscle and linking laminin (LN) in the extracellular matrix (ECM) to dystrophin in the submembraneous cytoskeleton. This dystrophin-associated glycoprotein complex (DGC) has been proposed to perform a structural role in skeletal muscle, its loss leading to loss of membrane integrity, muscle fiber degeneration and muscular dystrophy. alpha- and beta-DG appear to form the core of the DGC since alpha-DG is a high affinity LN receptor while beta-DG is a transmembrane protein that anchors alpha-DG to the membrane and interacts with dystrophin intracellularly. In order to determine the involvement of DG in skeletal muscle homeostasis and in LN assembly, we generated mouse muscle cell lines deficient in DG expression. Extensive characterization of these cells revealed that DG is essential for LN assembly on the surface of mature myotubes but that it is not involved in the maintenance of membrane integrity in culture. However, DG-deficient cells show increased apoptotic cell death during and after the period of myoblast differentiation into myotubes, indicating that DG is important for muscle cell survival. / The ECM molecule agrin has been implicated in the induction of acetylcholine receptor (AChR) aggregation at the neuromuscular junction (NMJ). The C-terminus of agrin shares significant homology with the region of LN that interacts with alpha-DG; we therefore reasoned that alpha-DG could be an agrin receptor. Ligand overlay assays revealed that alpha-DG binds agrin with high affinity and antibody perturbation experiments indicated that alpha-DG is involved in agrin-induced aggregation of AChRs. The role of alpha-DG in AChR aggregation was further studied using the DG deficient muscle cell lines. We found that alpha-DG is involved in the later stages of agrin-induced AChR aggregation. / We further localized DG and two of its intracellular ligands, dystrophin and its autosomal homologue utrophin, to a synaptic layer in the retina suggesting a role for DG in central nervous system synapses. DG, utrophin and LN are also co-expressed around blood vessels indicating a possible function in blood-brain barrier homeostasis.