Effects of Alternative Splicing of Capping Actin Protein (CAPZB) on Skeletal Muscle Cell Proliferation and Differentiation

The capping actin protein of muscle z-line subunit beta (CAPZB) is involved in the assembly and function of actin filaments in skeletal muscle cells. Exon of 113 nucleotides (nt) of CAPZB gene is regulated by alternative splicing. and the two protein isoforms are localized in different places within cardiac muscle cells. However, little is known about the localization or functions of the two isoforms in skeletal muscle cells. The first aim of this thesis was to better understand the functions of the two CAPZB splice isoforms in skeletal muscle cells. To address this question, we used morpholino antisense oligonucleotides to force the expression of the variant lacking the exon in C2C12 mouse myoblast cells and determine the impact of the two isoforms on cell proliferation and differentiation. The second goal of this thesis was to develop ImageJ macros for the automatic quantification of myotube area, total number of nuclei, and myotube fusion using microscopy images from immunofluorescence experiments. First, we found that both CAPZB splice isoforms behave similarly when evaluating muscle cell proliferation and differentiation. Second, when we compared the values achieved from the macros developed here with manual quantification done by researchers, we observed a high degree of correlation between the two methods of quantification. Therefore, the macros successfully developed in this thesis provided accurate biological values.Bachelor of Scienc

Development of a method for the determination of ultra-trace level mercury in adipose tissue by cold vapour atomic fluorescence spectrometry

A method for the determination of total mercury in rat adipose tissue by cold vapour atomic fluorescence spectrometry (CVAFS) has been developed. Adipose samples were initially subjected to a lyophilization procedure in order to facilitate the homogenization and accurate weighing of small tissue aliquots (~50 mg). A closed vessel microwave digestion procedure using a mixture of sulphuric and nitric acids was used to liberate mercury from the adipose matrix. All mercury species were quantitatively oxidized to Hg(II) by a potassium bromate/bromide oxidation, then reduced to Hg(0) vapour by stannous chloride prior to fluorescence detection. The CVAFS exhibited a linear range of 10 pg Hg/ml to 120 pg Hg/ml. The method detection limit in solution was 2 pg Hg/ml, or 1 ng Hg/g adipose tissue, based on a nominal 50 mg sample and a final volume of 25 ml. A reference material from the National Research Council of Canada (DOLT-2, trace metals in dogfish liver) was prepared in quadruplicate in order to assess the accuracy and precision of the method. Mercury in this material was recovered at 2.22 ± 0.08 μ g/g, which is 104% of the certified level (2.14 ± 0.10 μ g/g)

Power and limitations of electrophoretic separations in proteomics strategies

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed

Systematic comparison of the human saliva and plasma proteomes

The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 14474 unique peptide sequences identified from whole and ductal salivas[semi] 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61527/1/prca_200800140_sm_miscellaneous_information.pd