Bioexploration of wild Alaskan berries: From field screening to functional food

Wild berries are fundamental components of traditional diet and medicine for Native American and Alaska Native tribes and contain a diverse array of phytochemicals, including anthocyanins and proanthocyanidins, with known efficacy against metabolic disorders. Bioexploration represents a new paradigm under which bioactive preparations are screened in coordination with indigenous communities, to prepare for subsequent in-depth chemical and biological analysis. The inclusive, participatory philosophical approach utilized in bioexploration has additional benefits that could be realized in seemingly disparate areas, such as education and economics. Five species of wild Alaskan berries (Vaccinium uliginosum, V. ovalifolium, Empetrum nigrum, Rubus chamaemorus, and R. spectabilis) were tested using “Screens-to-Nature” (STN), a community-participatory approach to screen for potential bioactivity, in partnership with tribal members from three geographically distinct Alaskan villages: Akutan, Seldovia, and Point Hope. Berries were subsequently evaluated via HPLC and LC-MS2, yielding significant species and location-based variation in anthocyanins (0.9-438.6 mg eq /100g fw) and proanthocyanins (73.7-625.2 mg eq /100g fw). A-type proanthocyanidin dimers through tetramers were identified in all species tested. Berries were analyzed for in vitro and in vivo activity related to diabetes and obesity. R. spectabilis samples increased preadipocyte-factor-1 levels by 82% over control, and proanthocyanidin-rich fractions from multiple species reduced lipid accumulation in 3T3-L1 adipocytes. Furthermore, extracts of V. uliginosum and E. nigrum (Point Hope) reduced serum glucose levels in C57bl/6j mice up to 45%. The same precepts of bioexploration, especially the inclusion of indigenous community perspectives and knowledge, have relevance in other areas of study, such as education and economics. Studies have established the apathetic, low-motivational environment characteristic of many introductory science laboratory classes is detrimental to student interest, learning, and continuation in scientific education. A primary means of arresting this decline and stimulating the students’ attention and excitement is via engagement in hands-on experimentation and research. Using field workshops, the STN system is investigated as to its potential as a novel participatory educational tool, using assays centered around bioexploration and bioactive plant compounds that hold the potential to offset human health conditions. This evaluation of the STN system provided ample evidence as to its ability to augment and improve science education. Furthermore, Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis was employed as a theoretical framework to review the potential benefits and hurdles associated with developing a wild Alaskan berry commodity. Synthesizing various sources of information – including logistics and harvest costs, sources of initial capital, opportunities in the current superfruit industry, and socioeconomic factors – the development of a berry commodity proves to be a complex amalgam of competing factors which would require a delicate balance before proceeding.

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.