Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves 130-135).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 123-149.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 181 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-157).

Theses (Ph. D.)--Marshall University, 2004. / Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.

Thesis (M.S.)--Florida State University, 2006. / Advisor: Timothy S. Moerland, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Sept. 15, 2006). Document formatted into pages; contains x, 33 pages. Includes bibliographical references.

Thesis (M.S.)--University of Delaware, 2005. / Principal faculty advisor: Norman J. Karin, Dept. of Biological Sciences. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:28:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-14Bitstream added on 2014-06-13T20:08:43Z : No. of bitstreams: 1 000754514.pdf: 1170892 bytes, checksum: 2e07af1d94d3f5fbc4f47fea88dd613c (MD5) / O presente trabalho retrata estudo metaloproteômico no tecido muscular de bovinos da raça Nelore (Bos indicus) contrastantes para a característica de maciez da carne baseado em métodos de separação de proteínas por eletroforese bidimensional (2D-PAGE), identificação dos íons cálcio nos spots proteicos por fluorescência de raio-X (SR-XRF) e caracterização das proteínas por espectrometria de massas (ESI-MS). Os animais selecionados para compor o grupo M (carne macia) expressaram valores de força de cisalhamento (FC) entre 3,39 e 3,98 kg, já os animais selecionados para o grupo D (carne dura) expressaram valores de FC entre 7,11 e 7,45 kg. Foi incluído um terceiro grupo (P) de animais da raça Piemontês (Bos taurus) como modelo comparativo do grau de maciez da carne. O número médio de spots proteicos encontrados nas repetições dos géis dos grupos M, D e P foram de 186 ± 20, 146,5 ± 16,5 e 175 ± 15, respectivamente. As correlações obtidas nas repetições dos géis indicaram que os procedimentos de extração da proteína total foram eficientes e preservaram a estrutura metal-proteína. A maior detecção (56%) qualitativa de cálcio por SR-XRF nos spots proteicos de animais com carne macia é um indicativo da ocorrência da atividade proteolítica no tecido muscular durante o período post mortem. A 2D-PAGE foi eficiente no fracionamento das proteínas presentes em amostras de tecido muscular (Longissimus dorsi). As correlações obtidas nas repetições dos géis indicaram que os procedimentos de extração da proteína total foram eficientes e preservaram a estrutura metal-proteína / The present article describes a metalloproteomics study of bovine muscle tissue with different grades of meat tenderness from animals of the Nellore breed (Bos indicus) based on protein separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the identification of calcium ions in protein spots by Synchrotron Radiation X-ray Fluorescence (SR-XRF) and the characterization of proteins by electrospray ionization mass spectrometry (ESI-MS). The meat from the animals selected to form the Te group (tender meat) expressed shear force (SF) values ranging from 3.39 to 3.98 kg, and the meat from the animals selected for the To group (tough meat) expressed values ranging from SF 7.11 to 7.45 kg. A third group (P) of Piedmontese animals (Bos taurus) was included as a comparative model for the level of meat tenderness. The mean number of protein spots found in the gel replicates of the Te, To and P groups were 186 ± 20, 146.5 ± 16.5 and 175 ± 15, respectively. The correlations found in the gel replicates indicated that the total protein extraction procedures were efficient and preserved the metal-protein structure. The higher qualitative detection of calcium (56%) by SR-XRF in the protein spots of animals with tender meat was indicative of the occurrence of proteolytic activity in the muscle tissue during the post mortem period. The 2D-PAGE was efficient fractionation of proteins present in muscle tissue samples (Longissimus dorsi). The correlations obtained in repetitions of the gels indicated that the procedures for extraction of total protein were efficient and preserved the structure metal-protein

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.

The position of the cell division plane in plants is determined by the position of the preprophase band. The pre prophase band (PPB) is a ring of microtubules centered around the nucleus on the inner side of plasma membrane that establishes the cortical division site. The PPB forms at the end of G2 and breaks down at the end of prophase leaving behind protein markers of its position that are collectively called the cortical division site. During cytokinesis the phragmoplast expands towards the cortical division site and mediates the fusion of the new cell plate with the mother cell at that position. Several proteins necessary for PPB formation in plants have been identified, including maize DCD1 and ADD1 and Arabidopsis TON2, which are all type 2A protein phosphatase (PP2A)B" regulatory subunits. DCD1, ADD1, and TON2 localize to the PPB and the cortical division site through metaphase. The PP2A subunits each have two EF-hand domains, which are predicted to bind calcium ions. Since calcium ions are important for some aspects of cell division, we designed a series of constructs to test if TON2 binds calcium. TON2 protein was cloned into expression vectors, pET42a, and expression of TON2 protein was confirmed via Western blotting and immunodetection using a GST antibody. Site directed mutagenesis was used to mutate the TON2 EF-hand domains and mutated cDNAs were also cloned into expression vectors. These were then expressed in bacterial systems. Finally, the GST tagged proteins were purified. In the future, wild-type and mutated proteins TON2 proteins will used in calcium binding assays to determine if TON2 binds calcium.