Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

An examination of factors that influence entrepreneurial intention of high school students in Kenya

Abstract Considerable research has been carried out on entrepreneurship in efforts to understand its incidence in order to influence and maximize its benefits. Essentially, researchers and policy makers have sought to understand the link between individuals and business creation: Why some people start businesses while others do not. The research indicates that personality traits, individual background factors and association of entrepreneurship with career choice and small business enterprises, cannot sufficiently explain entrepreneurship. It is recognized that entrepreneurship is an intentional process and based on Ajzen’s Theory of Planned Behavior, the most defining characteristic of entrepreneurship is the intention to start a business. The purpose of this study was, therefore, to examine factors that influence entrepreneurial intention in high school students in Kenya. Specifically, the study aimed at determining if there were relationships between the perceptions of desirability, and feasibility of entrepreneurship with entrepreneurial intention of the students, identifying any difference in these perceptions with students of different backgrounds, and developing a model to predict entrepreneurship in the students. The study, therefore, tested how well Ajzen’s Theory of Planned Behavior applied in the Kenyan situation. A questionnaire was developed and administered to 969 final year high school students at a critical important point in their career decision making. Participants were selected using a combined convenience and random sampling technique, considering gender, rural/urban location, cost, and accessibility. Survey was the major method of data collection. Data analysis methods included descriptive statistics, correlation, ANOVA, factor analysis, effect size, and regression analysis. iii The findings of this study corroborate results from past studies. Attitudes are found to influence intention, and the attitudes to be moderated by individual background factors. Perceived personal desirability of entrepreneurship was found to have the greatest influence on entrepreneurial intention and perceived feasibility the lowest. The study findings also showed that perceived social desirability and feasibility of entrepreneurship contributed to perception of personal desirability, and that the background factors, including gender and prior experience, influenced entrepreneurial intention both directly and indirectly. In addition, based on the literature reviewed, the study finds that entrepreneurship promotion requires reduction of the high small business mortality rate and creation of both entrepreneurs and entrepreneurial opportunities (Kruger, 2000; Shane & Venkataraman, 2000). These findings have theoretical and practical implications for researchers, policy makers, teachers, and other entrepreneurship practitioners in Kenya.