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Globally, there are significant quantities of natural gas reserves that lie economically or physically stranded from markets. Options to monetize such reserves include Gas to Liquids (GTL) and Liquefied Natural Gas (LNG) technologies. GTL is a unique monetization option that brings natural gas products to crude oil markets. This technology is commercially immature, appears to have attractive market potential, requires substantial capital investments, and has uncertain operating costs and revenue generation. LNG is a more established monetization option. Project economics for the two technologies are reviewed, as well as literature evaluating such for either or both. Discounted cash flow models are studied for two project scenarios, and results are discussed and compared. The modeling effort seeks to inform the decision to invest in GTL or LNG for the monetization of a stranded gas reserve. / text

Success or failure of climate policies in limiting warming to beneath particular thresholds depends on several physical, economic and social uncertainties. Whilst scenario analysis can be informative as to the types of policies that are required to achieve these goals, the complexity of scenario analysis often masks the underlying fundamental choices. This dissertation introduces the concept of the ‘decarbonization identity' to simply and systematically describe the mutually exclusive and collectively exhaustive range of choices available in future climate policy decisions. The simple identity states that the remaining carbon budget [B] for a given level of warming can be partitioned into four areas: the already committed 'baked-in' emissions from existing capital stock [E]; new commitments arising from investments in additional capital stock yet to be made [N]; less the stranding of existing or future capital stock [S]; and the additional atmospheric space created by negative emissions technologies (NETs) [A]. This dissertation finds that currently operating electricity generators [E] would already emit more CO2 (~300 GtCO₂) then is compatible with currently available generation-only carbon budgets [B] for a temperature rise of 1.5-2°C (~240 GtCO₂). In addition, the current pipeline of planned fossil fuel power plants would add almost the same amount [N] of emission commitments (~270 GtCO₂) to this capital stock again. Finally, these carbon budgets are inherently uncertain and depend on future, yet to be achieved, reductions of short-lived climate pollutant (SLCP) emissions. Should those reductions not be achieved today's remaining carbon budgets could be up to 37% smaller. Policymakers have now five choices to achieve the Paris climate goals: (1) protect and enhance carbon budgets by early and decisive action on SLCPs; (2) retrofit existing power generators with carbon capture and storage (3) ensure that no new polluting capital stock is added; (4) strand a considerable amount of global electricity generation capacity; and (5) create additional atmospheric space by scaling up NETs. Over the coming years and decades, the challenge will be to identify the most efficient balance of these options.

Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious. Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.

Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed "beta" recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair. Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3' end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components. Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called bubble migration synthesis, a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms.

Homologous Recombination (HR) can promote genome stability through its capacity to faithfully repair DNA gouble 2trand !;!reak2 (DSBs) and preventing the demise of stalled replication forks in part by catalysing template switching to enable DNA polymerase to bypass lesions. Despite these beneficial roles, inappropriate or untimely HR events can have deleterious consequences. HR can cause genome instability by recombining "inappropriate" homologous sequences, especially if the recombination intermediates are resolved to form crossovers. Over the past few years, study of the rare inherited chromosome instability disorder, Eanconi Anaemia (FA), has uncovered a novel DNA damage response pathway. Although the FA pathway is required primarily for interstrand DNA cross link repair, its precise role in DNA repair reactions is still unclear. FA.Qomplementation group M (FANCM) is the sole component within the FA core complex which possesses a DNA helicase/ATPase domain and an endonuclease domain (albeit non-functional), suggesting that FANCM could translocate along DNA and target the FA core complex to blocked replication forks. To further elucidate the role of FANCM in HR, I have purified Fm11, the FANCM orthologue in the fission yeast Schizosaccharomyces pombe and tested its activity on a range of synthetic replication and recombination intermediates in vitro. Fml1 binds both replication forks and Holliday Junctions (HJs) which are key intermediates of HR.

Four strangers are stranded in an old farmhouse by a winter storm. Gilley lives on the farm. Shadows move of their own volition on the farm, and Gilley talks to echoes and sees the dead reflected in mirrors. Gilley's husband, Frank, disappeared over forty years ago. Jason is a college student who seeks Gilley out for an interview. He agrees to help Gilley find Frank. Jesse is a young boy who finds his way to the house after an accident. August is a private investigator whom Jason calls for help in finding Frank. August does not have a shadow nor a reflection of his own, and he can't remember how he lost them. Each wants something that only the others can provide, but each wants to keep their own secrets.

Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.