A sturdy house built on shifting soil: Separation of powers Interpretations from the bench

This thesis explores the development of the separation of powers doctrine and its application by the United States Supreme Court. Its analysis will focus upon the six different approaches to the doctrine that the Court has employed over the past two hundred years. Moreover, it will show that these six methods of analysis; which include the textual, original intent, structural, institutional competence, historical practice, and values approaches, have often been mixed single cases; The approach that is employed in these separation of powers cases often dictates their outcome. In many of these cases, a different approach may have led to a substantially different outcome. Thus, it will show that the doctrine has grown to have an ambiguous nature. This is both confusing and constraining to law makers and has led to the argument that the separation of powers doctrine no longer has a place in our modern political world

A Cell Engineering Approach to Enzyme-Based Fed-Batch Fermentation

One of the major disadvantages of batch fermentation is the difficulty in achieving high cell densities; in E.coli K12, much of this is attributed to the production of acetate via a phenomenon known as overflow metabolism. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch mechanisms require components which become problematic when applying them to smaller scale systems such as shake flasks. As a result, a number of slow release carbon techniques have been developed; one of which uses the enzymatic degradation of starch to slowly release glucose into the culture medium following the addition of an amylolytic enzyme. This reduces acetate production due to the low initial glucose concentration, leading to an increased cell density, and an increased product yield. To date, these amylolytic enzymes have been added to the culture exogenously, whereas this project aims to employ a cell engineering approach to design and build a self-secreting amylolytic chassis capable of enzyme-based fed-batch fermentation. The study explores the use of starch as an alternative carbon source, and describes the ability of a highly active amylolytic E. coli strain secreting S. thermoviolaceus α-amylase to degrade and utilise starch as a sole carbon source. Bacterial exo-acting amylolytic enzymes have been identified and cloned into E. coli for characterisation studies, with enhanced secretion of the novel C. violaceum glucoamylase using the DsbA signal peptide resulting in direct conversion of starch to glucose within the media. Further investigations reveal expression can be negatively regulated using a glucose sensitive promoter, providing a basis for self-regulation. Lastly, vectors have been constructed to simultaneously express the C. violaceum glucoamylase and a target recombinant protein (eGFP), resulting in higher biomass and increased recombinant protein expression when grown on starch compared to an equivalent amount of glucose, the first demonstration of a cell engineered approach to enzyme-based fed-batch fermentation

Dravidianism: Theorizing Identity, Religion, Culture, and Society in Tamil Reformist Thought

Around the turn of the 20th Century, a distinctive set of social and cultural reformist movements emerged in the Tamil-speaking region of Madras Presidency, one of the major administrative divisions of British colonial India. These movements, which were affiliated with thinkers such as Iyothee Thass, Maraimalai Adigal, and "Periyar" E.V. Ramasamy, articulated their reformist ideologies through the lens of a shared historical narrative that told the story of the "Aryan" or "Brahmin" subversion of an ancient and enlightened "Dravidian" or Tamil society indigenous to the Tamil South. This thesis argues that individual discursive terms such as "caste", "religion", "Tamil", and "Brahmin" in the "Dravidianist" discourse of these movements cannot be understood in isolation from each other; rather, they all participate in a semantic field that coheres around the core narrative of ancient Dravidian history

Application of the Implicit Association Test to a study on deception

Version of RecordThree experiments were conducted to find out whether the standard Implicit Association Test (IAT) could be used to distinguish truthful and deceitful witnesses. We anticipated that IAT effects would be greater after lying. Participants were asked to answer questions with incorrect answers (i.e., the lie condition) or correct answers (i.e., the truthful condition). A third group of participants were not interviewed (a control group). Participants then took the IAT, in which they were asked to associate correct and incorrect answers with positive or negative attributes. Results demonstrate that standard IAT effects are greater after lying than after truth telling, but only when attribute labels were clearly and explicitly linked to positive and negative affect. Theoretical implications are considered.Frost, P., Adie, M., Denomme, R., Lahaie, A., Sibley, A., & Smith, E.. (2010). Application of the Implicit Association Test to a Study on Deception. The American Journal of Psychology, 123(2), 221-230. http://www.jstor.org/stable/10.5406/amerjpsyc.123.2.022

Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development

BACKGROUND: Considerable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression. RESULTS: To profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo. CONCLUSIONS: Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-350) contains supplementary material, which is available to authorized users

Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii

BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth

Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker

Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR(r) and type 10 VAND-SNF(r). The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii

Toxoplasma gondii infection drives conversion of NK cells into ILC1-like cells

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here, we describ

Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection

Toxoplasma gondii is an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFNγ-mediated immunity to T. gondii infection is a complex, multifactorial process