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The hydro‑geomorphic structure and function of river and wetland systems provide a physical template for ecosystem functions, allowing for their recognition, conservation and rehabilitation. This thesis examines the diverse geomorphic structure and hydrological processes of a spectrum of chain‑of‑ponds morphologies, identifying the intrinsic and extrinsic controls on their formation, evolution and impairment or loss. Chains‑of‑ponds consist of steep‑sided ponds separated by densely vegetated alluvial valley‑fill sediments that contain shallow ephemeral channels or preferential flow paths. They are part of the spectrum of discontinuous watercourses that are important landscape features due to their unique geomorphological, hydrological and ecological function. Analysis of seven headwater chain‑of‑ponds reaches reveal morphological diversity, in both planform and stratigraphy, and in varying stages of evolution. Sensitivity assessments of these reaches, combined with two‑dimensional hydraulic modelling, precipitated the quantification of measurable pond and planform characteristics associated with geomorphic condition. This examination developed three quantitative metrics that provide a measure of event sensitivity, morphological sensitivity and change sensitivity that can be used to guide the management of smaller‑scale headwater systems. Stratigraphic analysis and luminescence dating of larger‑scale chains‑of‑ponds on higher‑order streams show different formation processes that result from threshold changes in fluvial energy over the late Quaternary, with antecedent controls on the size and position of much deeper (up to 7 m) and more stable ponds. This adds important insight to the range of non‑linear responses through both intrinsic controls and also more complex responses from the persistence of palaeo landforms. This analyses improve the understanding of intrinsic controls on fluvial evolution and will help assess recovery potential through forecasting future trajectories, guiding approaches to maintaining or restoring ecosystem function of discontinuous headwater systems in southeast Australia and similar systems internationally. Stable isotopes (δ18O and δ2H), 222Rn, and pond and floodplain water levels revealed that the hydrology and ecosystem function of large‑scale chains‑of‑ponds, and discontinuous fluvial systems more generally, could be highly sensitive to local‑scale water extraction, shallow groundwater aquifer interference and changes to groundwater recharge due to climatic changes. The surficial appearance of discontinuity masks the continuous longitudinal flow of the alluvial aquifer during both high‑flow and no‑flow stages, highlighting the importance of subsurface and hyporheic processes in defining the hydrological character of a river. This thesis strengthens the understanding of the hydro‑geomorphological processes of discontinuous systems to facilitate ecosystem managers to develop workable strategies for protecting and managing systems that, due to their discontinuous form, do not as yet receive the same legal status or protection as other rivers.

With advances in the sequencing and synthesis of DNA, automation, and computation, we are increasingly able to rapidly and reliably program functions into cells. However, because the functions we engineer cells to perform are often both unnecessary for the cell���s survival and burdensome to cell growth, mutation and natural selection can rapidly lead to loss of function. Though numerous strategies have made headway, improving the evolutionary stability of engineered functions remains a goal of the synthetic biology community. To address this problem generally, we developed a strategy relying on integrase-mediated recombination which allows non-producing progenitor cells to differentiate at a tunable rate, thereby continuously replenishing producer cells expressing the orthogonal T7 RNAP. While this strategy removes selective pressure for mutations inactivating the function of interest in the progenitor cell population, a strategy of terminal differentiation, in which the capacity of differentiated cells to grow is limited, was necessary to prevent the expansion of such mutations in the differentiated cell population. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate expression of T7 RNAP, and inactivate expression of �� protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. Critically, we demonstrated computationally that terminal differentiation endows the circuit with robustness to mutations which disrupt T7 RNAP driven expression, and to plasmid instability effects that result in decreased expression. Intuitively and computationally identifying the category of mutations which disrupt the differentiation process as the Achilles's heel of terminal differentiation, we developed a redundant architecture using a novel split-�� protein system which required 2 mutations to break the circuit. We experimentally demonstrated a trade-off between rate of production and duration of function as the differentiation rate is tuned, an increased benefit of terminal differentiation with higher-burden expression, and that redundancy improves the evolutionary stability of the terminal differentiation architecture. Specifically we achieve a maximum of ~2.8x (single-cassette terminal differentiation) and ~4.2x (redundant terminal differentiation) the total fluorescent protein production achieved from comparable high-burden naive expression in which all cells inducibly express T7 RNAP. We further demonstrate differentiation can enable the expression of even toxic functions, and develop a terminal differentiation circuit architecture which will allow the degree of redundancy and therefore the evolutionary stability of the architecture to be scaled to arbitrary degrees.

Background: Autophagy inhibition is a potential treatment for central nervous system (CNS) tumors. Autophagy, a heavily regulated process by which cellular waste is transferred to lysosomes for degradation and processing, is an integral part of tumor cell survival under stressful conditions including nutrient deprivation and chemotherapy. While the efficacy of autophagy inhibition has been demonstrated in CNS tumors with BRAF^(V600e) mutations, it has yet to be explored in other CNS tumor types with MAPK pathway dysregulation including NF1-mutated tumors. Many tumors associated with the NF1 phenotype can be difficult to treat surgically thus development of further pharmacologic interventions is necessary. Methods: A CRISPR/Cas9 mediated NF1 KO was derived from human immortalized Schwann cells and utilized as a tumor model. Autophagy inhibition was achieved pharmacologically by chloroquine (CQ) and genetically via shRNAi of ATG5 and ATG7. Trametinib was used for MEK inhibition. Cell growth and viability were determined by Incucyte, Cell Titer-Glo luminescent assay, and colony-formation assays. Protein expression was measured by western blot. Results: We demonstrate increased autophagic activity in NF1 KO cell as compared to control lines both at baseline and in response to cellular stress. Furthermore, we describe that NF1 KO cells exhibit increased sensitivity to CQ alone, CQ in combination with trametinib, and shRNAi-mediated autophagy inhibition in combination with trametinib. Conclusion: Here, we describe increased autophagic dependence of NF1 mutated tumors and demonstrate increased tumor sensitivity to autophagy inhibition both alone and in combination with MEK inhibition. These findings indicate that autophagy inhibition via CQ may be an effective adjunctive treatment for NF1 mutated tumors and suggests that diverse CNS tumor types with MAPK pathway dysregulation are susceptible to autophagy inhibition. Clinical investigation of combined MEK and autophagy inhibition has the potential to improve outcomes for NF1 patients with CNS tumors.

Application of synthetic biology is limited by the capacity of cells to faithfully execute burdensome engineered functions in the face of Darwinian evolution. Division of labor, both metabolic and reproductive, are underutilized in confronting this barrier. To address this, we developed a serine-integrase based differentiation circuit that allows control of the population composition through tuning of the differentiation rate and number of cell divisions differentiated cells can undergo. We applied this system to T7 RNAP-driven expression of a fluorescent protein, and demonstrate both increased duration of circuit function and total production for high burden expression. While T7 expression systems are typically used for high-level short-term expression, this system enables longer duration production, and could be readily applied to burdensome or toxic products not readily produced in bacteria.