Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Defining the Role of the Histone Methyltransferase, PR-Set7, in Maintaining the Genome Integrity of Drosophila Melanogaster

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

On the Regulation of Small Actors: Three Experimental Essays about Policies based on Voluntary Compliance and Decentralized Monitoring

Monitoring and enforcement are perhaps the biggest challenges in the design and implementation of environmental policies in developing countries where the actions of many small informal actors cause significant impacts on the ecosystem services and where the transaction costs for the state to regulate them could be enormous. This dissertation studies the potential of innovative institutions based on decentralized coordination and enforcement to induce better environmental outcomes. Such policies have in common that the state plays the role of providing the incentives for organization but the process of compliance happens through decentralized agreements, trust building, signaling and monitoring. I draw from the literatures in collective action, common-pool resources, game-theory and non-point source pollution to develop the instruments proposed here. To test the different conditions in which such policies could be implemented I designed two field-experiments that I conducted with small-scale gold miners in the Colombian Pacific and with users and providers of ecosystem services in the states of Veracruz, Quintana Roo and Yucatan in Mexico. This dissertation is organized in three essays.

The first essay, “Collective Incentives for Cleaner Small-Scale Gold Mining on the Frontier: Experimental Tests of Compliance with Group Incentives given Limited State Monitoring”, examines whether collective incentives, i.e. incentives provided to a group conditional on collective compliance, could “outsource” the required local monitoring, i.e. induce group interactions that extend the reach of the state that can observe only aggregate consequences in the context of small-scale gold mining. I employed a framed field-lab experiment in which the miners make decisions regarding mining intensity. The state sets a collective target for an environmental outcome, verifies compliance and provides a group reward for compliance which is split equally among members. Since the target set by the state transforms the situation into a coordination game, outcomes depend on expectations of what others will do. I conducted this experiment with 640 participants in a mining region of the Colombian Pacific and I examine different levels of policy severity and their ordering. The findings of the experiment suggest that such instruments can induce compliance but this regulation involves tradeoffs. For most severe targets – with rewards just above costs – raise gains if successful but can collapse rapidly and completely. In terms of group interactions, better outcomes are found when severity initially is lower suggesting learning.

The second essay, “Collective Compliance can be Efficient and Inequitable: Impacts of Leaders among Small-Scale Gold Miners in Colombia”, explores the channels through which communication help groups to coordinate in presence of collective incentives and whether the reached solutions are equitable or not. Also in the context of small-scale gold mining in the Colombian Pacific, I test the effect of communication in compliance with a collective environmental target. The results suggest that communication, as expected, helps to solve coordination challenges but still some groups reach agreements involving unequal outcomes. By examining the agreements that took place in each group, I observe that the main coordination mechanism was the presence of leaders that help other group members to clarify the situation. Interestingly, leaders not only helped groups to reach efficiency but also played a key role in equity by defining how the costs of compliance would be distributed among group members.

The third essay, “Creating Local PES Institutions and Increasing Impacts of PES in Mexico: A real-Time Watershed-Level Framed Field Experiment on Coordination and Conditionality”, considers the creation of a local payments for ecosystem services (PES) mechanism as an assurance game that requires the coordination between two groups of participants: upstream and downstream. Based on this assurance interaction, I explore the effect of allowing peer-sanctions on upstream behavior in the functioning of the mechanism. This field-lab experiment was implemented in three real cases of the Mexican Fondos Concurrentes (matching funds) program in the states of Veracruz, Quintana Roo and Yucatan, where 240 real users and 240 real providers of hydrological services were recruited and interacted with each other in real time. The experimental results suggest that initial trust-game behaviors align with participants’ perceptions and predicts baseline giving in assurance game. For upstream providers, i.e. those who get sanctioned, the threat and the use of sanctions increase contributions. Downstream users contribute less when offered the option to sanction – as if that option signal an uncooperative upstream – then the contributions rise in line with the complementarity in payments of the assurance game.