A plasmid-encoded Type III Secretion System (T3SS) is employed by human pathogenic yersiniae to inject effector proteins, termed Yops, directly into host cells. The secretion of Yops is tightly regulated, and occurs only upon contact with a eukaryotic cell in vivo or in media devoid of calcium in vitro. A complex containing the secreted protein YopN, its heterodimeric chaperone SycN/YscB, and TyeA is required to prevent secretion of effector Yops until the appropriate secretion-triggering signals are encountered. The mechanism by which these proteins regulate the T3S process is unknown. A mutational analysis of YopN and TyeA was performed to identify regions and residues of these proteins that are required to regulate Yop secretion. Amino-acid residues of TyeA were identified that were specifically required for the interaction of TyeA with YopN, confirming that the YopN/TyeA interaction is essential for the regulation of Yop secretion. Furthermore, analysis of TyeA mutants identified a surface-exposed region that was critical for the regulation of Yop secretion, but not required for interaction with YopN. YopN residues critical for the regulation of secretion clustered within the N- and C-terminal regions of YopN that were required to interact with the SycN/YscB chaperone and TyeA, respectively. No residues critical for the regulation of secretion were identified in the central region of YopN, suggesting that this region acts primarily to maintain proper positioning of the functional N- and C-terminal regions of this complex. A novel role for the chaperone binding domain (CBD) of YopN in the regulation of Yop secretion was identified. This role was separate from its role in binding the SycN/YscB chaperone and targeting YopN for secretion. Finally, it was demonstrated that the SycN/YscB chaperone is dispensable for the regulation of secretion if the expression of both YopN and TyeA is increased, indicating that these chaperones have no direct role in the regulation of Yop secretion. These results indicate that the YopN secretion signal and SycN/YscB chaperone function to efficiently target the YopN/TyeA complex to the T3S apparatus, whereas the YopN CBD and C-terminal region of YopN complexed with TyeA mediate the block in Yop secretion.

Swimming in ocean water, including beaches not impacted by known point sources of pollution, continues to pose a rising health concern. Studies which evaluate the relationship between indicator microbes, pathogens, and human health at recreational beaches are rare, especially at non-point beaches, although such studies are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this dissertation was to better understand the non-point source beach system, and hence determine possible directions that should be taken from a beach regulatory perspective in order to protect bather health and minimize economic burdens. This objective was accomplished through two studies. The first study included an initial evaluation of the presence of and associations among indicator microbes, pathogens and environmental conditions at a subtropical, recreational marine beach in South Florida (USA) impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events conducted at high or low tide and during elevated or reduced solar insolation conditions. Analyses included fecal indicator bacteria (FIB) (fecal coliform, Escherichia coli, enterococci, Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp. and Giardia spp.). The objective of the second study was to capture a series of "snapshots" of the study beach with respect to pathogens and indicator microbe levels, environmental conditions, and cumulative human health effects. Two sets of daily composite samples were collected during the BEACHES epidemiology study conducted in Miami (FL) at the same non-point source subtropical marine beach. These samples were analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters similar to those in the first study. Enterococci concentrations in water and sand assessed by qPCR were greater than membrane filtration measurements in both studies. FIB concentrations in water were below recreational water quality standards for three of four sampling events; pathogens and MST markers were also generally undetectable during the first study. FIB levels exceeded regulatory guidelines during one event, accompanied by the detection of HPyVs and pathogens, including the autochthonous bacterium Vibrio vulnificus in both sand and water, the allochthonous protozoan Giardia spp. in water, and Cryptosporidium spp. in sand samples. These elevated microbial levels were detected at high tide, low solar insolation conditions, and after elevated 2 day antecedent rainfall. Analyses of the second study demonstrated that rainfall and tide were also significant factors in determining the presence of both indicator microbes and pathogens. Additional potential associations between environmental factors, microbes and human health effects were also identified. However, more analyses should be conducted as the limited number of samples renders it difficult to make conclusive statements about the relationship between these different factors. The results of this research demonstrate the complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches will be needed to assess beach water quality for the purpose of protecting bather health. A recommended approach "The comprehensive tool box with approval process approach" to address the beach complexity is presented and applied to the study beach.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 1999. / Includes bibliographical references. Also available on the Internet.

Ph.D. / Molecular Biology / Neurospora crassa arg-2 gene encodes the small subunit of arginine specific carbamoyl phosphate synthethase, the first enzyme in fungal arginine (Arg) biosynthesis. The arg-2 mRNA contains an upstream open reading frame (uORF) specifying an evolutionarily conserved 24-residue peptide called the arginine attenuator peptide (AAP). Synthesis of the AAP causes ribosomes to stall on the mRNA in the presence of high concentrations of Arg. The amino acid sequence of the AAP, and not the sequence of its coding region, is responsible for this regulation. Scanning mutagenesis within the evolutionarily conserved region revealed that some residues are more important than others for the AAP to function. While most known nascent peptides that regulate translation are found encoded as uORFs or as N-terminal leader peptides, the AAP can exert regulatory function whether placed near the N-terminus or internally within a large polypeptide. The AAP’s peptide sensing features are conserved due to regulated stalling of fungal, plant, and animal ribosomes in response to Arg. That the AAP functions as an internal domain to regulate elongation in response to Arg establishes that such domains can provide a means of controlling translational elongation. The minimal sequences required for AAP to function as an internal domain was revealed by systematic deletion of its natural N- and C-terminal regions. Comparative analysis of the AAP with other fungi showed that the evolutionarily conserved region of the peptide is required for regulation. This minimal domain functions when placed as a uORF as seen by toeprint assay. Analyses of Arg analogs provided key insights in the structural requirements for Arg’s role in regulation. These results taken together provide a detailed picture of the requirements for Arg-specific regulation mediated by the AAP.

Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a fungal pathogen of maize that causes ear rots and stalk rots worldwide. In addition, it produces a group of mycotoxins called fumonisins when the fungus colonizes maize and maize-based products. Fumonisin B1 (FB1), the predominant form occurring in nature, can cause detrimental health effects in animals and humans. Several efforts were made to study the host and pathogen factors that contribute to the production of fumonisins. Using the available genomic resources, three genes with a potential role in FB1 regulation and development were identified. The genes are GBP1, GBB1 and GAP1. This research describes molecular characterization of these genes with respect to regulation of FB1 and development in F. verticillioides. GBP1 is a monomeric GTP binding protein with similarity to DRG and Obg sub-classes of G-proteins. GBB1 encodes heterotrimeric GTP binding protein β subunit. GAP1 is a GPI (Glycophosphotidylinositol) anchored protein, which belongs to a family of cell wall proteins. Targeted deletion and complementation studies indicated that GBP1 is negatively associated with FB1 biosynthesis but had no effect on conidiation in F. verticillioides. GBB1 plays an important role in regulation of FB1 biosynthesis, conidiation and hyphal growth, but not virulence. GAP1 is associated with growth, development and conidiation but not in positive regulation of FB1 or pathogenicity. The outcome of this study revealed new molecular genetic components that will help scientists better understand signal transduction pathways that regulate FB1 biosynthesis and conidiation in F. verticillioides.

Le système immunitaire nous protège contre les infections et les cancers. Cependant, des réponses immunitaires excessives ou incontrôlées peuvent causer des pathologies potentiellement mortelles comme le choc endotoxinique, des maladies auto-immunes, ou le rejet d’allogreffe. Une compréhension claire des mécanismes de régulation des réponses immunes permettrait d’envisager de nouvelles thérapeutiques plus ciblées. Dans ce travail réalisé chez la souris, la modulation de la réponse inflammatoire à une toxine bactérienne, le lipopolysaccharide (LPS), a été utilisée comme archétype de la régulation immunitaire. Ceci nous a permis de démontrer que la régulation de la prolifération homéostatique de lymphocytes T CD4+CD25- par des lymphocytes T régulateurs naturels tempère la réponse inflammatoire au LPS. Cet effet est notamment dépendant d’une diminution de la sécrétion d’IFN-γ par les lymphocytes activés par la prolifération induite par la lymphopénie. Nous avons aussi observé, que la désensibilisation du système immunitaire inné vis-à-vis du LPS, suite à des injections répétées d’endotoxine, induit le développement de cellules myéloïdes suppressives (myeloïd-derived suppressor cells MDSC) capables de réguler des réponses lymphocytaires T in vitro et in vivo. Nous avons pu mettre en évidence que l’effet suppresseur des MDSC est dépendant de leur expression de l’enzyme hème oxygénase-1 et de leur production d’IL-10. Le dialogue constant entre les cellules de l’immunité innée et de l’immunité acquise assure donc à la fois l’activation et la régulation du système immunitaire. Dans la majorité des cas, ceci permet aux réponses immunitaires d’être efficaces sans être excessives. La mise en évidence de ces processus identifie les lymphocytes T régulateurs et les cellules myéloïdes suppressives comme des éléments clefs de la régulation d’un processus inflammatoire. Les traitements immunosuppresseurs, les thérapies cellulaires et les greffes de moelle ont des effets variables et mal connu sur ces populations de cellules régulatrices. Tenir compte de ces interférences thérapeutiques avec les processus naturels de régulation de notre système immunitaire permettra certainement d’optimaliser l’utilisation de ce type de traitements. D’autre part, l’utilisation thérapeutique de ces deux types de cellules régulatrices pourrait être envisagé dans de nouvelles stratégies d’immuno-modulation plus « physiologique ».

For the past twenty years, environmental organizations have been the main force in Taiwan¡¦s environmental movement. Meanwhile, the institutionalization of environmental organizations had placed them in a dilemma of organizational sustaining and mission pursuing. To continue its influence on public policy and environmental awareness, environmental organizations need to perform harder and better. In recent years, the government has cast environmental organizations in more roles, as well as allowed them to play a more important role in policy consulting. Many environmental organizations have thus become intermediate social institutions and should be held accountable. Moreover, since all environmental organizations have ecology as its stakeholder and many organizations are advocacy-orientated, their process performance needs to be evaluated in the accountability procedure. From a self-regulation perspective, environmental organizations should also develop behavioral norms for self-governance to earn social recognition and trust. In practice, however, laws concerning the accountability of environmental organizations or other NPOs are not well formulated. The intent of this study is mainly to develop an accountability framework workable for self-assessment in environmental organizations. Taking a broad perspective, accountability is here defined as ¡§accounts for the results and process of organizational activities are provided, by which nonprofit organizations are responsive to expectations generated within and outside the organization so as to gain legitimacy in society.¡¨ Synthesizing large literature on theoretical, practical accountability and role expectancy on environmental organizations, the framework, based on institutional theory, was constructed to evaluate accountability in ¡§regulative¡¨, ¡§normative¡¨, ¡§cognitive¡¨ and ¡§organizational characteristics¡¨ dimensions, totally consisting of 92 items. Survey data from the leaders of environmental organizations were collected to examine the evaluative framework¡¦s feasibility by their assessing each item¡¦s reasonability, acceptability, practicability, and importance. As a result, 45 priority items out of 92 were selected, which identify the evaluative contents for an ¡§accountable¡¨ environmental organization, and can be taken as a useful checklist in future to scrutinize organizations¡¦ accountability and performance. Moreover, additional results from in-depth interviewing environmental organizations¡¦ leaders showed that they generally regard regulative accountability as acceptable, normative accountability as reasonable but difficult to achieve, and cognitive accountability as important in delineate organizations¡¦ mission and identity as well as adapting themselves to the society. Among the major findings of this studyare that environmental organizations accept their social responsibility and acknowledge the benefit of performance evaluation, but organizations and the society know little about the concept of accountability. These organizations also believed that the present social institutions and organizational conditions are unhelpful or even harmful for them to take accountability actions. These reasons incline environmental organizations to agree to more external regulation than self-regulation. In the meantime, the above situations mostly hinder launching accountability and performance evaluation in organizations. To drive self-regulation in environmental organizations, institutional building is suggested; that is to couple organizations¡¦ performance evaluation and advancement with self-assessment by expanding knowledge on accountability among the society and making institutional incentives available.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

The Mediator complex was originally discovered in the yeast Saccharomyces cerevisiae and has since then been shown to be required for transcriptional regulation both in vitro and in vivo. The Mediator complex also stimulates basal, unregulated transcription and serves as a bridge by conveying signals from promoter-bound transcriptional regulatory proteins such as activators and repressors to the RNA Polymerase II general transcriptional machinery. The Mediator consists of 21 subunits and can be divided into three distinct modules head, middle and tail. Despite the tremendous progress that has been achieved so far in characterizing the Mediator complex both functionally and structurally, many aspects of the complex are not yet well understood. The objective of this work is to achieve further understanding of the Mediator complex by studying the folding of different protein subunits, their interactions and how that affects assembly of the Mediator complex. In our first study we made a temperature-sensitive med21 mutant and used it to identify genes that can suppress the mutation when present in high copy number. Among the 10 genes that we identified, the strongest suppressors were Med7 and Med10, which encode Mediator subunits, and Ash1, which encodes a repressor of the HO gene. We also used 2-hybrid experiments and immunoprecipitation to study protein-protein interactions between Med21 and the Med4, Med7 and Med10 proteins which are all essential for viability and located within the middle domain of the Mediator complex. We found that the N-terminal 2-8 amino acids of Med21 are required for interactions with Med7 and Med10. These results led us to propose a model in which the N-terminal part of Med21 functions as a molecular switchboard where competing signals from various activators, repressors and mediator subunits are integrated prior to reaching the general transcription machinery. In our second study, we extended our studies of protein-protein interactions to another part of the mediator complex by studying the folding and the assembly processes of the mediator head domain subunits Med8, Med18 and Med20. Using purified proteins and a combination of several different methods such as immunoprecipitation, far-UV circular dichroism and fluorescence, we demonstrated that the Med8, Med18 and Med20 subunits are interdependent on each other for proper folding and complex formation.

Best execution obligations weigh on brokers when they execute orders to trade in shares for clients. These obligations have been seen as an outflow of general agency duties, and have been complemented by regulatory requirements related to best execution, dissemination of trading data, the handling of client orders and – in the United States – an obligation to execute at the best publicly available price or better (price protection). Here, different sets of real-world rules are analyzed with regard to transactional efficiency. Economic analyses are used to compare the effects of different rules, and are underpinned by a detailed analysis of relevant rules in the United States, the European Union, France, Sweden and England & Wales. Several normative conclusions can be drawn. Best execution rules that impose an agency duty on brokers do not seem to contribute in a discernible way to increased transactional efficiency. In contrast, disclosure rules that require both brokers and trading venues to provide ex post information about execution quality, and about how client orders have been routed, may contribute to mitigating the information asymmetry between brokers and clients. The compliance costs associated with such rules are outweighed by the positive effects on transactional efficiency. Lastly, a solution such as the US National Market System, which entails consolidated collection and dissemination of market data as well as price protection, can deliver significant efficiency gains through the virtual consolidation of trading venues. It also allows for more detailed regulations on different aspects of order execution, allow order execution regulations to function better, and is conducive to deeper integration of trading. However, creating such a system entails large initial investments. In the end, the choice whether to create a tightly-knit market system or not has wide-ranging implications for market structure, the design of regulatory rules and market integration.