Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references (p. 103-114).

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Murray V. Johnston, III, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

For the successful development of a hyperaccumulating plant sufficient for use in phytoremediation strategies, a thorough understanding of the mechanism of hyperaccumulation is required. A proteomic survey of the response of plants to metal exposure is a step towards this understanding. The frd3-3 metal accumulating mutant of Arabidopsis thaliana and its non-accumulating wildtype parental ecotype, Columbia, were grown hydroponically in growth chamber experiments and exposed to copper in the growth medium. The responses of the global and copper-targeted proteomes were examined both spatially and temporally. Exposure to copper caused a general increase in protein abundance, however, a prolonged exposure to copper that approached toxicity caused a decrease in protein abundance. The protein species differed between the roots of the two genotypes, with more defense- and stress-related proteins, and fewer transport and storage proteins identified in the mutant when compared to the wildtype. Proteomic evidence suggests that in the mutant the uptake and transport of copper ions to the aerial tissues is regulated. The protein expression patterns over time demonstrate a constitutive expression of defense- and stress-related proteins in the mutant, whereas the wildtype expression was one of induction. The constitutive expression of key defense proteins suggests a state-of-readiness for metal exposure in the mutant. The plant response to reactive oxygen species, as a consequence of copper exposure, is important in the overall metal accumulation mechanism. A suppression of the oxidative burst produced upon exposure to heavy metals is suggested by the proteomic evidence.

For the successful development of a hyperaccumulating plant sufficient for use in phytoremediation strategies, a thorough understanding of the mechanism of hyperaccumulation is required. A proteomic survey of the response of plants to metal exposure is a step towards this understanding. The frd3-3 metal accumulating mutant of Arabidopsis thaliana and its non-accumulating wildtype parental ecotype, Columbia, were grown hydroponically in growth chamber experiments and exposed to copper in the growth medium. The responses of the global and copper-targeted proteomes were examined both spatially and temporally. Exposure to copper caused a general increase in protein abundance, however, a prolonged exposure to copper that approached toxicity caused a decrease in protein abundance. The protein species differed between the roots of the two genotypes, with more defense- and stress-related proteins, and fewer transport and storage proteins identified in the mutant when compared to the wildtype. Proteomic evidence suggests that in the mutant the uptake and transport of copper ions to the aerial tissues is regulated. The protein expression patterns over time demonstrate a constitutive expression of defense- and stress-related proteins in the mutant, whereas the wildtype expression was one of induction. The constitutive expression of key defense proteins suggests a state-of-readiness for metal exposure in the mutant. The plant response to reactive oxygen species, as a consequence of copper exposure, is important in the overall metal accumulation mechanism. A suppression of the oxidative burst produced upon exposure to heavy metals is suggested by the proteomic evidence.

Hagfish are the plesiomorphic sister group of all vertebrates. They are scavengers and many live at depths reaching thousands of meters. In addition, hagfish show the lowest metabolic rate as well as cardiac performance in vertebrates. This dissertation evaluated the biochemical characterizations of hagfish skeletal muscles related to the feeding apparatus and hagfish cardiac muscle associated with cardiac performance and deep-sea effects at the proteomic and metabolomic levels. In Chapter one and two, I found creatine kinase over-expressed in hagfish somatic muscle and deep-sea hagfish cardiac muscle, I suppose that this enzyme was important for utilization of stored phosphocreatine in deep-sea animals¡¦ somatic muscle and cardiac muscle. Over-expressed glycogen phosphorylase in hagfish dental and deep-sea hagfish cardiac muscle supposes these two types of muscles undergoing the anaerobic glycolysis. Compared to teleosts (cobia and tuna), TMAO and urea were higher in hagfish suggest their functions in hagfish cardiac muscle as osmolytes, however, higher TMAO but not urea in deep-sea hagfish, I suggest TMAO functions not only as an osmolyte but also physiological impacts in hagfish cardiac muscle for depth-related adaptations. I also found higher nebulin express in hagfish cardiac muscle and higher tropomyosin express in cobia and tuna cardiac muscles, thus their contractile differentiations were resulted from the protein-protein mechanism. This dissertation provides candidate proteins and metabolits involved in ecophysiological adaptation of hagfish skeletal and cardiac muscles.

The swamp eel, Monopterus albus, is a benthic freshwater species, inhabiting the muddy ponds, canals, and rice fields. They are highly adaptive to stressful environment and, as a consequence M. albus is an aquaculture species. Under adverse environmental conditions, such as drought and high temperature, swamp eels burrow into the mud and enter into a stage of aestivation which is characterized by extremely slow physiological processes and complete quiescence. Reports on human muscle atrophy as a result of prolong lacking of muscle activity indicate that muscle atrophy is associated with reductions of number and sizes of muscle fibers. And this symptom is also called the disuse muscle atrophy. In this study, swamp eels were induced into aestivation by placing in a growth chamber under high temperature and low water contain in the mud substrate. Results show that when the swamp eel had been in aestivated for 100 days, the skeletal muscle atrophied about 48%. However, such ¡§atrophy¡¨ did not influence normal functions of the muscles, and the degree of atrophy much lower than non-aestivation species. The 2-DE results of the M. albus¡¦s muscle during 100 days of aestivation show that appearance or disappearance of new function proteins were not observed. However these were significant difference between three protein groups, including stress proteins, sarcomeric proteins and metabolic proteins. These three groups of proteins play important roles in prevention of atrophy of disused muscles. It is believe that M. albus is more suitable species than bear in the study of disuse muscle atrophy.