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Enterococcus faecalis is a Gram-positive bacterial species that is typically a member of the human gastrointestinal tract microbiota. However, E. faecalis is also a nosocomial pathogen, which is involved in urinary tract infections, soft tissue infections and endocarditis. In recent times, the occurrence of antibiotic resistance has complicated the treatment of these infections. One of the major differences between commensal and pathogenic strains of E. faecalis is that pathogens contain multiple mobile elements such as plasmids, transposons and integrative conjugative elements (ICE). These elements allow for the acquisition and transfer of virulence factors and resistance genes. Conjugative plasmids are a class of plasmids present in E. faecalis whose transfer to host cells is induced by a small pheromone peptide, cCF10 (LVTLVFV). This peptide is initially encoded as a 22-amino acid precursor (pre-cCF10) from the signal sequence of the chromosomal ccfA gene and is then proteolytically cleaved by signal peptidase II and Eep. Once pCF10 has been transferred a host E. faecalis cell, it is exceptionally stable. A replicon clone is maintained in greater than 85% of host cells over 100 generations in the absence of selection, suggesting the stability of pCF10 is intrinsic to the replicon. Three unique features of the replication initiation protein PrgW may contribute to this stability: (a) the interaction of PrgW with pre-cCF10, (b) disulfide bond formation at three conserved cysteines (C78, C275, and C307) in PrgW, and (c) processing of the nascent PrgW protein. Replication initiation proteins associated with theta replicons, such as pCF10, are often self-contained units. To initiate plasmid replication, the replication initiation protein (PrgW) binds to direct repeats (oriV) in its own coding sequence (prgW). In silico analysis of PrgW suggests the existence of three distinct domains within the protein. The first 122 amino acids are homologous to a conserved domain present in related replication initiation proteins, which includes a Helix-Turn-Helix (HTH) DNA binding domain. This suggests that this domain of PrgW has a DNA-binding function and binds to the oriV site in prgW. The following 61 amino acids are not similar to any known sequence, and are encoded by the DNA sequence containing the direct repeats in the oriV site. This domain may or may not have a distinct function. The remaining sequence forms a domain that contains cysteines C275 and C307, and is also similar to no known structure. It is hypothesized that this domain is related to the stability of pCF10. C307 appears to be critical, as previous experiments indicate that its mutation alone affects plasmid stability. Secondary structure analysis of this domain revealed the presence of multiple alpha-helices that contain distinct hydrophobic domains that possibly contribute to pre-cCF10 binding and PrgW tertiary structure. The positions of the conserved cysteines within these alpha-helices may stabilize a hydrophobic binding pocket that could potentially facilitate interaction with pre-cCF10. PrgW has a predicted molecular weight of 38.6 KDa and can be detected in Western blots as a band with an apparent approximate molecular weight (mw) of 36,000. Previous data from our lab indicates that, when overexpressed in E. faecalis, four bands of PrgW are present with observed molecular weights of 40,000, 36,000, 24,000 and 18,000. Time course experiments revealed that the 40,000 mw form is converted to a 36,000 mw form independent. The 40,000 mw form is unstable (with a complete turnover in 30 minutes) while the 36,000 mw form has a half-life of greater than 4 hours. The 24,000 mw band does not have a DNA binding motif and is likely a turnover product. When the three conserved cysteines (and only cysteines) in PrgW are replaced with alanine, the 40,000 mw form is still processed to the 36,000 mw form. However, the cysteine to alanine mutants accumulate the 36,000 mw form. / Microbiology and Immunology

Yes / In this article, we draw attention to public-private dilemmas among psychologists that make sense of the debates around the replication crisis, citation practices and networking practices. We argue that the values of justice and caring map onto the public sphere and private sphere respectively and create the horns of a dilemma for academics. While bureaucratic justice is a publicly revered value of modernity in psychological research that underpins ethics, validity, reliability and equality of opportunity, ‘caring’ is a more subtle value of traditionalism that runs in parallel and is detected only by our psychological practices. In particular, we argue that it is detected by practices such as disputes between the replicated and their replicators in replication studies (understood as a dramatic counter reality) as to who is more ‘careless’ with procedure; citation (including the self-care of self-citation) as thanksgiving and incantation of powerful others in enchantment rituals, and the system of professional indebtedness that accrues in ‘kinship’ networks – where kinship is understood broadly as adoption into a research group and its network. The clashes between these values can lead to a sense of hypocrisy and irony in academic life, as incommensurate values split between private and public expression. From this position, we delve into Isaiah Berlin's work on incommensurate values to suggest that ethical pluralism, involving more public recognition of the equal but different ethical demands of these values can help overcome these everyday dilemmas in the public sphere.

State machine replication (SMR) is one of the most widely studied and used methodology for building highly available distributed applications and services. SMR replicates a service across a set of computing hosts, and executes client operations on the replicas in an agreed- upon total order, ensuring linearizability of the replicated shared state. The problem of determining a total order reduces to one of computing consensus. State-of-the-art consensus protocols are inadequate for newer classes of applications such as Blockchains and for geographically distributed infrastructures. The widely used Crash Fault Tolerance (CFT) fault model of consensus protocols is prone to malicious and adversarial behaviors as well as non-crash faults such as software bugs. The Byzantine fault-tolerance (BFT) model and its trust-based variant, the hybrid model, permit stronger failure adversaries. However, state-of-the-art Byzantine and hybrid consensus protocols have performance limitations in geographically distributed environments: they designate a primary replica for proposing total-orders, which becomes a bottleneck and yields sub-optimal latencies for faraway clients. Additionally, they do not scale to hundreds of replicas and provide consistent performance as the system size grows. To overcome these limitations and develop highly scalable SMR solutions, this dissertation presents two leaderless consensus protocols, namely ezBFT and Dester, for the Byzantine and hybrid models, respectively. These protocols enable every replica to receive and order client commands. Additionally, they exchange command dependencies to collectively order commands without relying on a primary. Our experimental evaluations in a 7-node geographically distributed setup reveals that ezBFT improves client-side latency by as much as 40% over state-of-the-art BFT protocols including PBFT, FaB, and Zyzzyva. Dester, for the hybrid model, reduces latency by as much as 30% over ezBFT. Next, the dissertation presents a new paradigm called DQBFT for designing consensus protocols that can scale to hundreds of nodes in geographically distributed environments. Since leaderless protocols exchange command dependencies, they do not scale to hundreds of nodes. DQBFT overcomes this scalability limitation by decentralizing only the heavy task of replicating commands and centralizing the process of ordering the commands. While DQBFT can be used to enhance existing primary-based protocols, Destiny is a hybrid instantiation of the DQBFT paradigm using linear communication for better scalability than naive instantiations. Experimental evaluations in a 193-node geographically distributed setup reveal that Destiny achieves ≈ 3× better throughput and ≈50% better latency than state-of-the-art BFT protocols including Hotstuff, SBFT, and Hybster. Lastly, the dissertation presents two techniques for designing and implementing BFT protocols with reduced development costs. The dissertation presents Bumblebee, a methodology for manually transforming CFT protocols to tolerate Byzantine faults using trusted execution environments that are increasingly available in commodity hardware. Bumblebee is based on the observation that CFT protocols are incapable of tolerating non-malicous non-crash faults, but they are nevertheless deployed in many production systems. Bumblebee provides a Generic Algorithm that can represent protocols in both CFT and hybrid fault models, thus allowing easy construction of hybrid protocols using CFT protocols as baselines. The dissertation constructs hybrid instantiations of CFT protocols including Paxos, Raft, and M2Paxos. Experimental evaluations of the hybrid variants reveal that they perform at par with native hybrid protocols, but incur a 30% overhead over their CFT counterparts. Hybrid protocols rely on the integrity of trusted execution environments, which are increasingly subject to security exploits. To withstand exploits, the dissertation presents DuoBFT, a protocol that exposes both the BFT and hybrid fault models within a single consensus protocol. This enables consensus under both fault models within the same protocol and without additional redundancy, allowing DuoBFT to achieve the performance of hybrid protocols and the security of BFT protocols. Experimental evaluations reveal that DuoBFT achieves the best of both hybrid and BFT fault models with less than 10% overhead. / Doctor of Philosophy / Computers are ubiquitous; they perform some of the most complex and safety-critical tasks such as controlling aircraft, managing the financial markets, and maintaining sensitive medical records. The undeniable fact is that computers are faulty. They are prone to crash and can behave arbitrarily. Even the most robust computers such as those that are sent to the outer space eventually fail. External phenomenon such as power outages and network disruptions affect their operation. To make computing systems reliable, researchers and practitioners have long focussed on interconnecting many individual computers and programming them to effectively be duplicates of one another. This way when one computer fails in a system, the rest of the computers still ensure that the system as a whole is operational. Duplication requires that multiple computers effectively perform the same task. In order for multiple computers to perform the same task together, they should first agree on the task. More generally, since computing systems perform multiple tasks, they should agree on the sequence of tasks that they will individually perform and follow the agreement. This is what is known as the State Machine Replication technique. State Machine Replication (SMR) is a powerful technique that is applicable to numerous computing applications. Blockchain systems, the technology behind the cryptocurrencies such as Bitcoin and Ethereum, uses the SMR technique. In the context of Blockchain, the added challenge in that some of the computers involved in SMR can be programmed by adversarial parties and could act in a way to jeopardize the integrity of the whole system. For Bitcoin and Ethereum, this could mean embezzlement of hundreds or even millions of dollars worth of cryptocurrencies. Certain SMR systems are capable of tolerating such intrusions and ensure system integrity. Such systems are deemed to be Byzantine tolerant. This dissertation presents designs, techniques, and implementations of Byzantine State Machine Replication systems. The problems addressed in this dissertation are those that plague existing Byzantine SMR systems making them suboptimal for newer applications such as Blockchains. First, when computers that participate in SMR are spread around the world, their performance is dependent on the communication latencies between any two pair of computers. Second, the number of computers required is proportional the number of adversarial computers that need to be tolerated. Consequently, certain SMR systems for Blockchains require hundreds of computers to tolerate heavy adversarial behavior. Many existing SMR technique perform poorly under these scenarios. The techniques presented in this dissertation address various permutations of these challenges.

DRAM contributes significantly to a server system's cost and global warming potential. To make matters worse, DRAM density scaling has not kept up with the scaling in logic and storage technologies. An effective way to reduce DRAM's monetary and environmental cost is to increase its effective utilization and extract the best possible performance in all utilization scenarios. To this end, this dissertation proposes Utilization-adaptive Memory Architectures that enhance the memory controller with the ability to adapt to current memory utilization and implement techniques to boost system performance. These techniques fall under two categories: (i) The techniques under Utilization-adaptive Hardware Memory Replication target the scenario where memory is underutilized and aim to boost performance versus a conventional system without replication, and (ii) The techniques under Utilization-adaptive Hardware Memory Compression target the scenario where memory utilization is high and aim to significantly increase memory capacity while closing the performance gap versus a conventional system that has sufficient memory and does not require compression. / Doctor of Philosophy / A computer system's memory stores information for the system's immediate use (e.g., data and instructions for in-use programs). The performance and capacity of the dominant memory technology – Dynamic Random Access Memory (DRAM) – has not kept up with advancements in computing devices such as CPUs. Furthermore, DRAM significantly contributes to a server's carbon footprint because a server can have over a thousand DRAM chips – substantially more than any other type of chip. DRAM's manufacturing cycle and lifetime energy use make it the most carbon-unfriendly component on today's servers. To reduce the environmental impact of DRAM, an intuitive way is to increase its utilization. To this end, this dissertation explores Utilization-adaptive Memory Architectures which enable the memory controller to adapt to the system's current memory through a variety of techniques such as: (i) Utilization-adaptive Hardware Memory Replication which copies in-use data to free memory and uses the extra copy to improve performance, and (ii) Utilization-adaptive Hardware Memory Compression which uses dense representation for data to save memory and allows the system to run applications that require more memory than the physically installed memory. Compared to conventional systems that do not feature these techniques, these techniques improve performance for different memory utilization scenarios ranging from low to high.

Despite being one of the most well-known laboratory-based tasks in psychology, certain methodological and theoretical considerations surrounding the marshmallow test have gone largely unstudied until recently. These considerations reflect gaps in the delay of gratification literature and broader contemporary issues concerning the replicability of seminal findings and the lack of an agreed upon theoretical framework in the field. Accordingly, my dissertation uses the classic marshmallow test to explore the contemporary issues of replication and theory in psychology in a series of three studies. In Study 1, the marshmallow test is at the center of a case study unpacking the nuances of direct and conceptual replication; a tool designed to support ongoing replication efforts is proposed. Study 2 executes a full-scale replication of the paradigm from the case study, and introduces a methodological extension to improve the paradigm’s experimental rigour while making it amenable to an evolutionary–developmental framework. Finally, Study 3 applies an evolutionary–developmental framework to examine how this perspective might help account for individual differences in marshmallow test behavior. Through these three studies, my dissertation provides an example of how engaging in replication and applying an evolutionary–developmental framework to the marshmallow test literature to inform outstanding theoretical questions in psychology might be mutually beneficial endeavors. / Thesis / Doctor of Philosophy (PhD) / This dissertation uses an iconic measure in psychology, “the marshmallow test,” to explore issues currently relevant to the field today. The marshmallow test measures delay of gratification––how children chose between a small reward now or a larger reward later. However, despite being one of the most well-known laboratory-based tasks in psychology, certain methodological and theoretical considerations surrounding the marshmallow test have gone largely unstudied until recently. These considerations reflect two bigger issues relevant to psychology more broadly: that of how well findings in psychology can be replicated, and the lack of an overarching and unifying theory in the field. Using the marshmallow test, this dissertation 1) proposes a tool to support ongoing replication efforts, 2) executes a replication and extension of a recent well-cited study, and 3) introduces ways of exploring how a framework that takes evolutionary–developmental principles into account might help address outstanding theoretical questions in the study of delay of gratification.

<p>To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. </p><p>DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in <italic> Drosophila Menalogaster </italic>, is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4<super>Ddb1</super> mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase. </p><p>In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.</p><p>In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.</p> / Dissertation