Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

Compreendendo a utopia realizável: uma defesa do ideal de justiça distributiva da teoria de John Rawls

O trabalho tem por objetivo articular uma defesa da teoria de justiça distributiva de John Rawls, considerando-se, para tal, as mudanças que o autor efetuou em sua teoria de justiça como equidade. Assim, a pesquisa tomará como base não somente o critério de justiça distributiva que se consolidou em Uma Teoria de Justiça, através do princípio da diferença, mas também avaliará de que forma este ideal continua presente nos textos posteriores do autor: O Liberalismo Político e O Direito dos Povos. Para tal, o estudo retomará as críticas cosmopolitas à proposta de internacionalização da teoria de justiça como equidade e, à luz destas, apresentará uma defesa do projeto de Rawls, evidenciando elementos do mesmo que estão alinhados ao projeto de justiça distributiva e sugerindo que sua proposta teórica é coerente com as premissas de Uma Teoria de Justiça, apesar de o princípio da diferença não estar presente entre os princípios fundamentais que devem ser estabelecidos entre os povos. Logo, trata-se também de uma proposta interpretativa que se vincula à compreensão (minoritária) de que as mudanças teóricas efetuadas por Rawls em sua teoria não a tornaram incompatível com seu projeto originário estabelecido em Uma Teoria de Justiça.

Assessment of contaminant body burdens and histopathology of fish and shellfish species frequently used for subsistence food by Alaskan Native communities

Subsistence food items can be a health concern in rural Alaska because community members often rely on fish and wildlife resources not routinely monitored for persistent bioaccumulative contaminants and pathogens. Subsistence activities are a large part of the traditional culture, as well as a means of providing protein in the diets for Tribal members. In response to the growing concerns among Native communities, contaminant body burden and histopathological condition of chum and sockeye salmon (Oncorhynchus keta and Oncorhynchus nerka) and the shellfish cockles and softshell clams (Clinocardium nuttallii and Mya arenaria) were assessed. In the Spring of 2010, the fish and shellfish were collected from traditional subsistence harvest areas in the vicinity of Nanwalek, Port Graham, and Seldovia, AK, and were analyzed for trace metals and residues of organic contaminants routinely monitored by the NOAA National Status & Trends Program (NS&T). Additionally, the fish and shellfish were histologically characterized for the presence, prevalence and severity of tissue pathology, disease, and parasite infection. The fish and shellfish sampled showed low tissue contamination, and pathologic effects of the parasites and diseases were absent or minimal. Taken together, the results showed that the fish and shellfish were healthy and pose no safety concern for consumption. This study provides reliable chemistry and histopathology information for local resource managers and Alaska Native people regarding subsistence fish and shellfish use and management needs.

Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell line

Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genescytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3 beta-hydroxysteroid dehydrogenase (3PHSD2), 17 beta-hydroxysteroid dehydrogenase (17 beta HSD1 and 17 beta HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3PHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption.

Enhancement of quality factor for TE whispering-gallery modes in microcylinder resonators

The enhancement of quality factor for TE whispering-gallery modes is analyzed for three-dimensional microcylinder resonators based on the destructive interference between vertical leakage modes. In the microcylinder resonator, the TE whispering-gallery modes can couple with vertical propagation modes, which results in vertical radiation loss and low quality factors. However, the vertical loss can be canceled by choosing appropriate thickness of the upper cladding layer or radius of the microcylinder. A mode quality factor increase by three orders of magnitude is predicted by finite-difference time-domain simulation. Furthermore, the condition of vertical leakage cancellation is analyzed.

Choices of approaches in e-moderation: conclusions from a grounded theory study

Much has been published in recent years about the desirable nature of facilitated interactions in on-line discussions with educational purposes. However little has been reported about the roles which tutors actually adopt in real life learning contexts, how these range between ‘tutoring, ‘managing’ and ‘facilitating’, and what the distinctions between these three roles may be. In this paper choices of priorities in e-moderation, which were made in three naturalistic (real life) case studies by three higher education practitioners, are identified and discussed. These contrasting approaches were captured and analysed using grounded theory principles. The paper also discusses these occasions when the facilitation was less effective than might have been desired. It finally summarises the potential of various approaches within e-moderation – and some of the attendant risks. The finding is that principles and practices developed for face-to-face support of student-directed learning were found equally applicable in e-moderated online group work, despite several significant differences between the two types of setting. Keywords: higher education, e-learning, e-moderation, asynchronous discussions, learning outcomes, grounded theory

Robust Identification of Shared Losses Using End-to-End Unicast Probes

Current Internet transport protocols make end-to-end measurements and maintain per-connection state to regulate the use of shared network resources. When two or more such connections share a common endpoint, there is an opportunity to correlate the end-to-end measurements made by these protocols to better diagnose and control the use of shared resources. We develop packet probing techniques to determine whether a pair of connections experience shared congestion. Correct, efficient diagnoses could enable new techniques for aggregate congestion control, QoS admission control, connection scheduling and mirror site selection. Our extensive simulation results demonstrate that the conditional (Bayesian) probing approach we employ provides superior accuracy, converges faster, and tolerates a wider range of network conditions than recently proposed memoryless (Markovian) probing approaches for addressing this opportunity.

Shear effects on the properties and separation characteristics of whey protein precipitates

Selective isoelectric whey protein precipitation and aggregation is carried out at laboratory scale in a standard configuration batch agitation vessel. Geometric scale-up of this operation is implemented on the basis of constant impeller power input per unit volume and subsequent clarification is achieved by high speed disc-stack centrifugation. Particle size and fractal geometry are important in achieving efficient separation while aggregates need to be strong enough to resist the more extreme levels of shear that are encountered during processing, for example through pumps, valves and at the centrifuge inlet zone. This study investigates how impeller agitation intensity and ageing time affect aggregate size, strength, fractal dimension and hindered settling rate at laboratory scale in order to determine conditions conducive for improved separation. Particle strength is measured by observing the effects of subjecting aggregates to moderate and high levels of process shear in a capillary rig and through a partially open ball-valve respectively. The protein precipitate yield is also investigated with respect to ageing time and impeller agitation intensity. A pilot scale study is undertaken to investigate scale-up and how agitation vessel shear affects centrifugal separation efficiency. Laboratory scale studies show that precipitates subject to higher impeller shear-rates during the addition of the precipitation agent are smaller but more compact than those subject to lower impeller agitation and are better able to resist turbulent breakage. They are thus more likely to provide a better feed for more efficient centrifugal separation. Protein precipitation yield improves significantly with ageing, and 50 minutes of ageing is required to obtain a 70 - 80% yield of α-lactalbumin. Geometric scale-up of the agitation vessel at constant power per unit volume results in aggregates of broadly similar size exhibiting similar trends but with some differences due to the absence of dynamic similarity due to longer circulation time and higher tip speed in the larger vessel. Disc stack centrifuge clarification efficiency curves show aggregates formed at higher shear-rates separate more efficiently, in accordance with laboratory scale projections. Exposure of aggregates to highly turbulent conditions, even for short exposure times, can lead to a large reduction in particle size. Thus, improving separation efficiencies can be achieved by the identification of high shear zones in a centrifugal process and the subsequent elimination or amelioration of such.

Mathematical modelling and optimisation of the formulation and manufacture of aggregate food products

In this PhD study, mathematical modelling and optimisation of granola production has been carried out. Granola is an aggregated food product used in breakfast cereals and cereal bars. It is a baked crispy food product typically incorporating oats, other cereals and nuts bound together with a binder, such as honey, water and oil, to form a structured unit aggregate. In this work, the design and operation of two parallel processes to produce aggregate granola products were incorporated: i) a high shear mixing granulation stage (in a designated granulator) followed by drying/toasting in an oven. ii) a continuous fluidised bed followed by drying/toasting in an oven. In addition, the particle breakage of granola during pneumatic conveying produced by both a high shear granulator (HSG) and fluidised bed granulator (FBG) process were examined. Products were pneumatically conveyed in a purpose built conveying rig designed to mimic product conveying and packaging. Three different conveying rig configurations were employed; a straight pipe, a rig consisting two 45° bends and one with 90° bend. It was observed that the least amount of breakage occurred in the straight pipe while the most breakage occurred at 90° bend pipe. Moreover, lower levels of breakage were observed in two 45° bend pipe than the 90° bend vi pipe configuration. In general, increasing the impact angle increases the degree of breakage. Additionally for the granules produced in the HSG, those produced at 300 rpm have the lowest breakage rates while the granules produced at 150 rpm have the highest breakage rates. This effect clearly the importance of shear history (during granule production) on breakage rates during subsequent processing. In terms of the FBG there was no single operating parameter that was deemed to have a significant effect on breakage during subsequent conveying. A population balance model was developed to analyse the particle breakage occurring during pneumatic conveying. The population balance equations that govern this breakage process are solved using discretization. The Markov chain method was used for the solution of PBEs for this process. This study found that increasing the air velocity (by increasing the air pressure to the rig), results in increased breakage among granola aggregates. Furthermore, the analysis carried out in this work provides that a greater degree of breakage of granola aggregates occur in line with an increase in bend angle.

Soft sediment ecology of Lough Hyne Marine Reserve - spatial and temporal patterns

Distribution of soft sediment benthic fauna and the environmental factors affecting them were studied, to investigate changes across spatial and temporal scales. Investigations took place at Lough Hyne Marine Reserve using a range of methods. Data on the sedimentation rates of organic and inorganic matter were collected at monthly intervals for one year at a number of sites around the Lough, by use of vertical midwater-column sediment traps. Sedimentation of these two fractions were not coupled; inorganic matter sedimentation depended on hydrodynamic and weather factors, while the organic matter sedimentation was more complex, being dependent on biological and chemical processes in the water column. The effects of regular hypoxic episodes on benthic fauna due to a natural seasonal thermocline were studied in the deep Western Trough, using camera-equipped remotely-operated vehicle to follow transects, on a three-monthly basis over one year. In late summer, the area below the thermocline of the Western Trough was devoid of visible fauna. Decapod crustaceans were the first taxon to make use of ameliorating oxygen conditions in autumn, by darting below the thermocline depth, most likely to scavenge. This was indicated by tracks that they left on the surface of the Trough floor. Some species, most noticeably Fries’ goby Lesueurigobius friesii, migrated below the thermocline depth when conditions were normoxic and established semi-permanent burrows. Their population encompassed all size classes, indicating that this habitat was not limited to juveniles of this territorial species. Recolonisation by macrofauna and burrowing megafauna was studied during normoxic conditions, from November 2009 to May 2010. Macrofauna displayed a typical post-disturbance pattern of recolonisation with one species, the polychaete Scalibregma inflatum, occurring at high abundance levels in March 2010. In May, this population had become significantly reduced and a more diverse community was established. The abundance of burrowing infauna comprising decapods crabs and Fries’ gobies, was estimated by identifying and counting their distinctive burrow structures. While above the summer thermocline depth, burrow abundance increased in a linear fashion, below the thermocline depth a slight reduction of burrow abundance occurred in May, when oxygen conditions deteriorated again. The majority of the burrows occurring in May were made by Fries’ gobies, which are thought to encounter low oxygen concentrations in their burrows. Reduction in burrow abundance of burrowing shrimps Calocaris macandreae and Callianassa subterranea (based on descriptions of burrow structures from the literature), from March to May, might be related to their reduced activity in hypoxia, leading to loss of structural burrow maintenance. Spatial and temporal changes to macrofaunal assemblage structures were studied seasonally for one year across 5 sites in the Lough and subject to multivariate statistical analysis. Assemblage structures were significantly correlated with organic matter levels in the sediment, the amounts of organic matter settling out of the water column one month before macrofaunal sampling took place as well as current speed and temperature. This study was the first to investigate patterns and processes in the Lough soft sediment ecology across all 3 basins on a temporal and spatial scale. An investigation into the oceanographic aspects of the development, behaviour and break-down of the summer thermocline of Lough Hyne was performed in collaboration with researchers from other Irish institutions.

TEACHING AMERICAN CIVIL WAR MUSIC IDSTORY WITH MODERN EDITIONS OF PERIOD MUSIC FOR FULL CONCERT BAND

This dissertation explores a method of teaching the history of Civil War music and musicians through modern full-band editions of original brass band music. In the study of music history the period of the Civil War is rarely discussed, or at best, mentioned only if a student takes a specific course on the history of bands and happens to look deeply into the background of some of the early band pioneers such as Patrick Gilmore, who served in the Union Army as a bandmaster. The history of the musicians, bands, and music performed during the Civil War deserves study to provide a way for students and audiences to learn this history. This project includes lesson plans that can be used with the arrangements of the period music as well as select published music that is also representative of the period. Included with the historical information are four arrangements of original brass band music now scored for full concert band. Each arrangement includes a section scored for brass only with optional brass band parts. Historical information is provided on the Civil War period bands and how each side used them, on the composers of the music, and also on the individual compositions. The historical information can be used to supplement the lesson plans to teach the history, as well as for program notes for audiences. The research involved locating information on both Union and Confederate bands available in books, other dissertations, articles, and interviews with Civil War music historians. The original brass band music is scored for full band. This method will allow teachers and conductors to highlight this period of wind band history and to share it with both students and audiences. Included with this project are photos and video footage taken during a visit with the 1st Brigade Band of Watertown, Wisconsin, an historical organization dedicated to recreating the music and performances of an actual Civil War era band.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.