Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Fertility Associated Antigen (FAA) produced by the accessory sex glands and contained within the seminal fluid binds heparin and facilitates capacitation in ejaculated sperm, resulting in improved fertility in bulls capable of producing the protein. In this study, a total of 206 bulls derived from three populations were evaluated for the presence or absence of FAA through utilization of the Repro Test at three semen collections over a 60-d period. Across all collections, the percentage of FAA Negative bulls ranged from 13.64 to 36.11%. Within the three populations, 32, 33, and 67 bulls were observed at three different collections, of which 3.03, 9.09 and 4.48% were FAA Negative at all three collections, respectively. Furthermore, 27.27, 33.33, and 20.90% of bulls were observed to have variations within their FAA status after providing an initial FAA Positive result, respectively. Bull age, sperm concentration, progressive forward motility, percent normal sperm, ejaculate volume, and scrotal circumference were determined to be significantly different between FAA Negative and FAA Positive bulls in at least one collection. However, no consistent trend was observed across populations, or collections within a population, with regard to a relationship between these variables and FAA. Furthermore, of fourteen bulls that produced an ejaculate in which no sperm was detected, 78.57% (n=11) were FAA Positive despite the lack of sperm within the ejaculate. No single variable commonly measured to determine bull fertility was consistent in predicting the FAA status of bulls. The ability to produce FAA precedes puberty and the Repro Test can be used to identify FAA in prepuberal bulls. However, a large percentage of bulls, both prepuberal and peripuberal, are capable of displaying variation in their FAA status (as determined by the Repro Test) over time.

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

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--Northern Michigan University, 2009. / Includes bibliographical references (leaves 65-73).

CD4+ lymphocytes (T helper cells) play a crucial role in orchestrating adaptive immune responses. This project aims to characterise the CD4+ cells in zebrafish (Danio rerio) for a better understanding of adaptive immunity in teleost fish. I cloned three CD4 homologues, termed zfCD4 and zfCD4 related (zfCD4rel1 and zfCD4rel2), with both reported previously in other bony fish species. The zfCD4 and zfCD4rels transcripts were detected in immune organs in zebrafish and were most highly expressed in the lymphocyte population. A moderate induction of the zfCD4 and zfCD4rels (and other immune related genes) was seen in kidney, spleen and intestine after poly (I:C) injection. Two antibodies against CD4 and IFN-γ, respectively, have been validated using various immunostaining approaches for further functional studies. The CD4 positive cells ranged from 20-30% of lymphocytes in zebrafish, similar to what is seen in other vertebrates. The expression level of IFN-γ and other Th cell related genes were analysed in immunised fish following re-stimulation with antigen, revealing that in zfCD4+ lymphocytes an increased expression of cytokines and master transcript factors was seen when the same antigen was used for boosting. This is the first demonstration of Th-type responses effected by CD4+ lymphocytes in a poikilotherm. Lastly, I studied an aspect of Treg function in zebrafish, focused on a master transcription factor of Tregs, the FoxP3 gene. Knocking down FoxP3 genes in zebrafish resulted in modulated gene expression of cytokines and transcription factors associated with Th and Treg cells, providing some evidence that the immune tolerance function of Treg cells may exist in teleost fish, with some sub-functionalisation of the two FoxP3 paralogues apparent. This thesis extends our knowledge into teleost adaptive immune responses mediated by CD4+ Th cells and putative FoxP3+ Treg cells and may aid future studies using zebrafish as a model of vertebrate immune function.

Recombinant antibodies have increased in prominence as therapeutics and diagnostic tools since their introduction to the market in the mid-1980s. They are used to treat diverse conditions from Crohn's disease to cancer. Since the Anthrax letter attacks of 2001, a great deal of work has been carried out to develop therapeutics to this disease, and antibodies that neutralize the toxic action of Bacillus anthracis are prominent among them. This dissertation describes the elucidation of the structure of the 14B7 family of neutralizing antibodies directed at protective antigen (PA) of B. anthracis and the complex of PA domain 4 (PAD4) with an ultra-high affinity neutralizing antibody (M18), and then utilizes this information to aid in the engineering of the antibody to various ends. Chapter 2 presents the structure of the M18-PAD4 complex and of the 14B7 family of antibodies, which aids in the understanding of the affinity maturation process for this antibody family. Chapter 3 describes the affinity maturation of M18 to a PA variant by applying the knowledge gained from the complex structure. This previously intractable challenge was met by employing saturation mutagenesis in highly focused libraries to M18 directed by the complex structure to the area of variation on PA. These results indicate that this could be a generalizable method for the engineering of M18 to natural and deliberate variation of PA. Chapter 4 reports work toward the development of a reversible, photoresponsive antibody using small molecule and polymer-protein conjugates. The results indicate that a probable site on M18 was located for placement of the polymer appendage, although further work is necessary to empirically refine the properties of the photoresponsive polymer. Chapter 5 presents an unrelated project, which was to confirm the existence of a proposed RNA thermosensor in the 5' untranslated region of LcrF from the pathogenic bacterium Yersinia pestis, the causative agent of plague. Overall, these studies reveal the power of structure-based engineering in this antibody-antigen system. In addition, the structural elucidation of the M18-PAD4 complex and the 14B7 family of antibodies furthers our basic understanding of protein-protein interactions and the process of affinity maturation of antibodies. / text

published_or_final_version / abstract / toc / Biochemistry / Doctoral / Doctor of Philosophy

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