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The mechanism of non-genotoxic hepatocarcinogenicity of the antihistaminic drug methapyrilene was studied using rat hepatocytes. Optimal survival of rat hepatocytes in culture was obtained when cells were plated in serum containing (10% FCS) medium, left to attach for 2 hours and then maintained in serum-free medium containing EGF, insulin and dexamethasone. At 48 hours of culture there was maximal stimulation of DNA synthesis by EGF and 0.5 μM methapyrilene and 1 mM phenobarbital stimulated DNA synthesis further. Methapyrilene was demonstrated to be an uncoupler of respiration as it produced a concentration-dependent increase in State 4 respiration rate of isolated rat hepatocytes. Low concentrations of MP produced stimulation of mitochondrial membrane potential and elevation of intracellular Ca2+ levels when isolated rat hepatocytes were incubated with methapyrilene for 1 to 5 minutes. Prolonged exposure to methapyrilene or high concentrations (3-10 mM) produced a toxic effect, inhibiting mitochondrial membrane potential and inducing cell death. The effect of methapyrilene on programmed cell death (apoptosis) was studied with a fluorescent dye (Hoechst 33342), which stained DNA. The number of apoptotic cells with condensed chromatin was counted in hepatocytes cultured in medium with and without TGF-β1 and several concentrations of test chemical. Methapyrilene did not induce apoptosis in serum-containing medium, whereas in the presence of EGF and hormones apoptosis was induced. The effect of methapyrilene on apoptosis was similar to the effect of phenobarbitone and it was dependent on culture conditions (presence of serum or EGF and hormones). Methapyrilene produced an increase in release of intracellular Ca2+ into the culture medium after 24 hours in culture and a dose-dependent decrease in triglyceride content after 48 hours in culture. The mechanism of hepatocarcinogenicity of methapyrilene can be explained by the modulation of cell proliferation and apoptosis through signal transduction pathways. Intracellular Ca2+ would be one of the second messengers involved as an increase in intracellular Ca2+ precedes both cell proliferation and apoptosis. An optimal method of cryopreserving hepatocytes was investigated. Cooling rates between -10 and -50 C/min appear optimal. The temperature of release of heat of fusion was the critical point in the cryopreservation protocols. Rapid cooling rates which reduced the release of heat of fusion were optimal. Hepatocytes after cryopreservation maintained similar levels of DNA synthesis to freshly prepared cultures and it was also stimulated by EGF after 48 hours in culture. However, Neutral Red uptake was decreased and cryopreserved hepatocytes were more sensitive to the toxic effect of methapyrilene. Cryopreservation had an adverse effect on mitochondrial function because there were more cells with lower mitochondrial membrane potential compared to freshly isolated cells and an impairment of the respiration function was demonstrated.

Cells adapt their metabolism to satisfy changing bioenergetic and biosynthetic needs. Investigation of metabolic reprogramming in cancer has provided insight into the metabolic control of proliferation and survival. While the predominant focus of this field has been aerobic glycolysis (the Warburg effect), increasing evidence points to a complex landscape of tumor metabolic circuitries beyond aerobic glycolysis, including varied degrees of mitochondrial contribution to tumor metabolism. To investigate alternative metabolic programs compatible with tumor growth, we turned to Diffuse Large B-cell Lymphoma (DLBCL), a highly heterogeneous disease encompassing discrete clusters or subtypes defined by tumor-intrinsic genetic distinctions. In one classification scheme, a B-cell receptor (BCR)/proliferation cluster identified BCR-dependent DLBCLs with elevated expression of BCR signaling components. A second subset, OxPhos-DLBCL, displayed increased expression of mitochondrial oxidative phosphorylation genes, and was insensitive to BCR inhibition. However, the functional attributes of OxPhos-DLBCLs and the nature of their BCR-independent survival were unknown. Upon integrative analyses of DLBCL subtypes, we uncovered quantitative proteome- and metabolome-level signatures associated with differences in nutrient and energy metabolism. Specifically, BCR-DLBCLs have greater glycolytic flux typical of the Warburg phenotype. Unlike BCR-DLBCLs, OxPhos-DLBCLs channel the majority of glucose-derived pyruvate into mitochondria, display elevated mitochondrial electron transport chain (ETC) activity, ATP production, and fatty acid oxidation (FAO). Importantly, these metabolic distinctions are associated with subtype-selective survival mechanisms. Moreover, acute inhibition of BCR signaling in BCR-DLBCLs increased their FAO capacity, thus revealing a reciprocal relationship between BCR and FAO. Further dissection of mitochondrial function in OxPhos-DLBCLs indicates that increased mitochondrial metabolism is integrated with at least two homeostatic mechanisms that help maintain ETC activity and FAO capacity. In particular, OxPhos-DLBCLs harbor robust protein-level enrichment of mitochondrial translation factors required for the synthesis of mitochondrial-DNA-encoded ETC subunits. Inhibition of the mitochondrial translation pathway is selectively toxic to OxPhos-DLBCLs. A second mitochondrial homeostatic pathway, mitochondrial network dynamics, also proved relevant to OxPhos-DLBCLs. Compared to BCR-DLBCLs, OxPhos-DLBCLs display a fragmented mitochondrial network that supports their FAO capacity. Overall, these findings demonstrate previously unappreciated metabolic heterogeneity in molecular subsets of DLBCL and uncover BCR-independent survival mechanisms linked to mitochondrial FAO, protein translation, and network architecture.

Communities of color and low-socioeconomic status are disproportionately impacted by food, energy, and water insecurity (Hoff, 2011). Additionally, these communities have been historically left out of environmental movements and policy making. Thus, it is particularly important to involve youth of marginalized identities in these issues. However, school learning is often disconnected from students’ sociocultural and sociohistorical backgrounds and experiences (Buxton, 2010; Ladson-Billings, 2014; Paris & Alim, 2014). Community-based education (CBE) is one way to provide learning experiences contextualized in real-world issues, that happen in socioculturally relevant ways, and that support young people in contributing meaningfully to their lives and communities. In three distinct, but connected studies, this dissertation explores the opportunities and challenges in community-based education; in preparing university mentors for this work, and in developing and sustaining a community-university partnership. The research in this dissertation is situated within the context of Growing North Minneapolis (GNM) - a partnership between the North Minneapolis community and the University of Minnesota (UMN) that runs an urban environmental and agriculture internship program for youth of marginalized identities. First, an explanatory, single embedded case design was used to examine the ways in which community-based education through internships can support youth who are marginalized in traditional educational environments. The experiences of youth in four different garden groups are explored at length. This study highlights the holistic educational opportunities found in community-based environments, particularly bringing attention to the value of intergenerational mentoring and work, diversity in learning communities, and the value of work-based learning. Second, the focus transitions from the youth to the university mentors. Specifically, an explanatory, single case study design was employed to examine the growth in critical mentoring capacity of the university mentors during their community-based mentoring experience and to consider how university mentors can be prepared to work with communities and youth of marginalized identities in socioculturally relevant ways. Findings from this study outline themes in the knowledge and dispositions that university mentors developed during their preparation course and their internship experience. Key recommendations are offered to prepare mentors of dominant and privileged identities for mentoring work with youth and community of marginalized identities. Third, the evolution of the program partnership between the community and the university is explored. A design-based research (DBR) approach was utilized to examine the ways in which community and university knowledge, skills, and perspectives coevolved, clashed and coexisted over the first three years of the partnership. This study details three design iterations, focusing on the interplay of community and university knowledge and skills, challenges, and growth. This study brings attention to the importance of leveraging diverse community and university perspectives, epistemologies, knowledge, skills, resources, and types of capital in harmony to support communities and young people who have been historically underserved and systemically marginalized. Collectively, the three studies featured in this dissertation have important implications for community-based learning for youth of marginalized identities; preparing university mentors of dominant identities for this work; and the opportunities in community-university partnerships to strengthen this work.