published_or_final_version / abstract / Ecology and Biodiversity / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Cellular proliferation is a key characteristic of hematopoietic stem and progenitor cells (HSC/HPCs) that allows for the production of all blood cell lineages during an individuals lifetime. While this feature of stem cells is strictly regulated during steadystate and stress hematopoiesis, it also contributes to the development of myeloproliferative disorders, such as chronic myelogenous leukemia, essential thrombocythemia, and polycythemia vera. It should come as no surprise then, that common treatments for these diseases often target the proliferative nature of the dysfunctional HSC/HPCs. Thus, the identification of molecular determinants of cell cycle regulation associated with these disorders could serve as targets for novel therapies. Using the hematopoietic system of the inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), it was found that the HSC/HPCs of the long-lived B6 mouse strain were less susceptible to the cytostatic agent hydroxyurea (HU) than the short-lived D2 mouse strain. A quantitative trait locus (QTL) analysis revealed a region of proximal chromosome 7 that regulates this response to HU. Congenic mouse strains were generated and phenotypic analysis confirmed that the B6 and D2 loci confer a low and high sensitivity of the HSC/HPCs to HU, respectively. We then showed that while this response of the HSC/HPCs to HU is independent of their cell cycle status, the B6 allele of this QTL confers a proliferative advantage to bone marrow cells after bone marrow transplantation. Having shown that proximal chromosome 7 regulates the response of HSC/HPCs to HU, we found it necessary to characterize the gene and protein expression profiles in order to identify the responsible candidate genes. We first analyzed mRNA expression profiles of HPCs from the parental and congenic mouse strains using gene microarrays and found that four genes within the congenic interval were differentially expressed. Real-time PCR confirmed that the expression profile of only one gene, Ndufa3, is significantly different in HPCs of B6 and D2 mice. Concurrently, we assessed the protein expression profiles of HPC-enriched mononuclear cells. Significant differences were found between the cytoplasmic and nuclear fractions of both strains, with a skewing of protein expression towards the D2 congenic strain.

The recent emergence of the field of proteomics has been driven by advances in mass spectrometry methods and instrumentation. Due to the large amount of data generated, success at peptide and protein identification is contingent on reliable software algorithms. The software programs in use at the time the work in this dissertation was carried out were well suited to the task of identifying unmodified peptides and proteins in complex mixtures. However, the existing programs were not able to reliably identify protein modifications, especially unpredicted modifications. This dissertation describes the development of two novel software algorithms that can be used to screen LC-MS-MS data files, and identify MS-MS spectra that correspond to peptides with either predicted or unpredicted modifications. The first program, SALSA, is highly flexible and uses user defined search criteria to screen data files for spectra the exhibit fragmentation patterns diagnostic of specific modifications or peptide sequences. SALSA facilitates exhaustive searches, but requires user expertise to both generate search criteria and to validate matched spectra. The second program, P-Mod, provides automated searches for spectra corresponding to peptides in a search list. P-Mod is able to identify spectra derived from either modified or unmodified peptides. All sequence-to-spectrum matches reported in the P-Mod output are assigned statistical confidence levels derived using extreme value statistics.

Mass spectrometry plays a significant role in many proteomics experiments owing to its ability to provide high quality, detailed data on complex samples containing proteins and/or their constituent peptides. As with any technology, the capabilities of mass spectrometers are constantly increasing to provide better resolution, faster data acquisition, and more accurate mass measurements. However, the existence and widespread use of previous-generation instruments is not negligible. While these instruments may not have the capabilities of their modern counterparts they are still able to collect useful experimental data, though their limitations can result in trade-offs between certain parameters such as resolution, sample run-time, and tandem MS experiments. This work describes an alternative method of MS data analysis, dubbed Parallel Isotopic Tag Screening (PITS), which seeks to enable higher throughput and the collection of better quality data on such previous generation instruments.

The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells.

No description available.