No description available.

No description available.

The Hippo signaling pathway offers an intrinsic mechanism to control organ sizes, and dysfunction of this pathway can often lead to cancer. Great advancement has been made in recent years into understanding this pathway. Despite all this invaluable knowledge, much remains to be explored. Mass spectrometry offers an unbiased approach to characterize the interactome of any protein of interest and is particularly powerful for identifying potential novel regulators of signalling pathways. I therefore set out to characterize the interactome of all the Hippo pathway main components using mass spectrometry, with the goal of uncovering novel regulatory mechanism(s) of the Hippo pathway. In the end, I was able to identify over 250 novel interactors of the Hippo pathway in total. This study demonstrates the utility of mass spectrometry to identify novel regulators of the Hippo pathway and characterization of one such interactor.

Breast cancer is a major health issue, affecting annually approximately 1.4 million women worldwide. It is a highly heterogeneous disease with the different subtypes having distinct clinical outcomes and different sensitivity to various treatment modalities. The focus of the present dissertation was the identification of novel proteomic prognostic markers for patients with early stage breast cancer. Three different approaches to identify potential prognostic markers were undertaken. First, we hypothesized that since different breast cancer subtypes have distinct clinical outcomes, breast cancer subtype-specific proteins may retain prognostic potential. Second, given the central role of estrogen signaling in breast epithelial cell biology, we hypothesized that estrogen-regulated proteins may be useful in predicting patient outcome. Finally, we hypothesized that genes related to survival based on meta-analyses of publicly available breast cancer tissue microarray data, may also demonstrate prognostic potential at the proteome level. As such, a variety of mass spectrometry-based approaches and biological samples were utilized for the discovery of these potential prognostic protein markers resulting in twenty-four candidates. Upon the identification of candidate biomarkers, a mass spectrometry-based assay for the simultaneous quantification of these proteins in breast cancer tissue samples was established. The developed assay was used for measuring the relative expression levels of the potential biomarkers in a cohort of 96 breast cancer tissue samples from untreated patients with early stage breast cancer. This exercise uncovered two proteins that showed the potential to discriminate between ER-positive patients at high and low risk of disease recurrence, namely KPNA2 and CDK1. In conclusion, the present dissertation describes the development of a preclinical exploratory study, from the discovery to the preliminary verification of potential prognostic biomarkers for breast cancer patients.

No description available.

Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.

v, 245 leaves ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1974) from the Dept. of Organic Chemistry, University of Adelaide

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 220-237).

Thesis (Ph. D.)--University of Akron, Dept. of Chemistry, 2006. / "December, 2006." Title from electronic dissertation title page (viewed 04/24/2008). Advisor, Chrys Wesdemiotis; Committee members, Matthew P. Espe, Jun Hu, Wiley J. Youngs, Frank W. Harris; Department Chair, Kim C. Calvo; Dean of the College, Ronald F. Levant; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.