Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Signal transduction, regulation, and developmental logic of EGFR signalling in C. elegans

RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.

My research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.

In an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.

By comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.

Finally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.

The Mechanical Genome in Regulation and Infection

Biological information storage and retrieval is a dynamic process that requires the genome to undergo dramatic structural rearrangements. Recent advances in single-molecule techniques have allowed precise quantification of the nano-mechanical properties of DNA [1, 2], and direct in vivo observation of molecules in action [3]. In this work, we will examine elasticity in protein-mediated DNA looping, whose structural rearrangement is essential for transcriptional regulation in both prokaryotes and eukaryotes. We will look at hydrodynamics in the process of viral DNA ejection, which mediates information transfer and exchange and has prominent implications in evolution. As in the case of Kepler's laws of planetary motion leading to Newton's gravitational theory, and the allometric scaling laws in biology revealing the organizing principles of complex networks [4], experimental data collapse in these biological phenomena has guided much of our studies and urged us to find the underlying physical principles.

Optimization and Control of Power Flow in Distribution Networks

Climate change is arguably the most critical issue facing our generation and the next. As we move towards a sustainable future, the grid is rapidly evolving with the integration of more and more renewable energy resources and the emergence of electric vehicles. In particular, large scale adoption of residential and commercial solar photovoltaics (PV) plants is completely changing the traditional slowly-varying unidirectional power flow nature of distribution systems. High share of intermittent renewables pose several technical challenges, including voltage and frequency control. But along with these challenges, renewable generators also bring with them millions of new DC-AC inverter controllers each year. These fast power electronic devices can provide an unprecedented opportunity to increase energy efficiency and improve power quality, if combined with well-designed inverter control algorithms. The main goal of this dissertation is to develop scalable power flow optimization and control methods that achieve system-wide efficiency, reliability, and robustness for power distribution networks of future with high penetration of distributed inverter-based renewable generators.

Proposed solutions to power flow control problems in the literature range from fully centralized to fully local ones. In this thesis, we will focus on the two ends of this spectrum. In the first half of this thesis (chapters 2 and 3), we seek optimal solutions to voltage control problems provided a centralized architecture with complete information. These solutions are particularly important for better understanding the overall system behavior and can serve as a benchmark to compare the performance of other control methods against. To this end, we first propose a branch flow model (BFM) for the analysis and optimization of radial and meshed networks. This model leads to a new approach to solve optimal power flow (OPF) problems using a two step relaxation procedure, which has proven to be both reliable and computationally efficient in dealing with the non-convexity of power flow equations in radial and weakly-meshed distribution networks. We will then apply the results to fast time- scale inverter var control problem and evaluate the performance on real-world circuits in Southern California Edison’s service territory.

The second half (chapters 4 and 5), however, is dedicated to study local control approaches, as they are the only options available for immediate implementation on today’s distribution networks that lack sufficient monitoring and communication infrastructure. In particular, we will follow a reverse and forward engineering approach to study the recently proposed piecewise linear volt/var control curves. It is the aim of this dissertation to tackle some key problems in these two areas and contribute by providing rigorous theoretical basis for future work.