Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells

Electrostatic Contributions to the Thermodynamics of Ribonuclease P Protein Folding

Electrostatic interactions are of fundamental importance in determining the structure and stability of macromolecules. For example, charge-charge interactions modulate the folding and binding of proteins and influence protein solubility. Electrostatic interactions are highly variable and can be both favorable and unfavorable. The ability to quantify these interactions is challenging but vital to understanding the detailed balance and major roles that they have in different proteins and biological processes. Measuring pKa values of ionizable groups provides a sensitive method for experimentally probing the electrostatic properties of a protein.

pKa values report the free energy of site-specific proton binding and provide a direct means of studying protein folding and pH-dependent stability. Using a combination of NMR, circular dichroism, and fluorescence spectroscopy along with singular value decomposition, we investigated the contributions of electrostatic interactions to the thermodynamic stability and folding of the protein subunit of Bacillus subtilis ribonuclease P, P protein. Taken together, the results suggest that unfavorable electrostatics alone do not account for the fact that P protein is intrinsically unfolded in the absence of ligand because the pKa differences observed between the folded and unfolded state are small. Presumably, multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

Detrital layer thickness in 12 varved sediment cores in Lake Mondsee and related flood events AD1976-2005

A succession of 23 sub-millimetre to maximum 12-mm-thick, mostly flood-triggered detrital layers, deposited between 1976 and 2005, was analysed in 12 varved surface sediment cores from meso-scale peri-alpine Lake Mondsee applying microfacies and high-resolution micro X-ray fluorescence analyses. Detailed intrabasin comparison of these layers enabled identification of (i) different source areas of detrital sediments, (ii) flood-triggered sediment flux and local erosion events, and (iii) seasonal differences of suspended flood sediment distribution within the lake basin. Additional calibration of the detrital layer record with river discharge and precipitation data reveals different empirical thresholds for flood layer deposition for different parts of the basin. At proximal locations detrital layer deposition requires floods exceeding a daily discharge of 40 m**3/s, whereas at a location 2 km more distal an hourly discharge of 80 m**3/s and at least 2 days of discharge above 40 m**3/s are necessary. Furthermore, we observe a better correlation between layer thickness and flood amplitude in the depocentre than in distal and proximal areas of the basin. Although our results are partly site-specific, the applied dual calibration approach is suitable to precisely decipher flood layer formation processes and, thereby, improve the interpretation of long flood time series from lake sediments.

Foraminiferal assemblage and statistical analysis of surface sediment samples from the Golfe d'Arguin, Mauritania

With the growing pressure of eutrophication in tropical regions, the Mauritian shelf provides a natural situation to understand the variability in mesotrophic assemblages. Site-specific dynamics occur throughout the 1200 m depth gradient. The shallow assemblages divide into three types of warm-water mesotrophic foraminiferal assemblages, which is not only a consequence of high primary productivity restricting light to the benthos but due to low pore water oxygenation, shelf geomorphology, and sediment partitioning. In the intermediate depth (approx. 500 m), the increase in foraminiferal diversity is due to the cold-water coral habitat providing a greater range of micro niches. Planktonic species characterise the lower bathyal zone, which emphasizes the reduced benthic carbonate production at depth. Although, due to the strong hydrodynamics within the Golf, planktonic species occur in notable abundances through out the whole depth gradient. Overall, this study can easily be compared to other tropical marine settings investigating the long-term effects of tropical eutrophication and the biogeographic distribution of carbonate producing organisms.

Mg/Ca and Sr/Ca in calcites and estimated distribution coefficients in marine sediments (Table 2)

Recrystallization processes in marine sediments can alter the extent to which biogenic calcite composition serves as a proxy of oceanic chemical and isotopic history. Models of calcite recrystallization developed to date have resulted in significant insights into these processes, but are not completely adequate to describe the conditions of recrystallization. Marine sediments frequently have concentration gradients in interstitial dissolved calcium, magnesium, and strontium which have probably evolved during sediment accumulation. Realistic, albeit simplified, models of the temporal evolution of interstitial water profiles of Ca, Mg, and Sr were used with several patterns of recrystallization rate variation to predict the composition of recrystallized inorganic calcite. Comparison of predictions with measured Mg/Ca and Sr/Ca ratios in severely altered calcite samples from several Deep Sea Drilling Project sites demonstrates that models incorporating temporal variation in interstitial water composition more successfully predict observed calcite compositions than do models which rely solely on present-day interstitial water chemistry. Temporal changes in interstitial composition are particularly important in interpreting Mg/Ca ratios in conjunction with Sr/Ca ratios. Estimates of Mg distribution coefficients from previous observations in marine sediments, much lower than those in laboratory studies of inorganic calcite, are confirmed by these results. Evaluation of the effects of diagenetic alteration of biogenic calcium carbonate sediment must be a site-specific process, taking into account accumulation history, present interstitial chemistry and its variation in the past, and sample depths and ages.

Statistically robust representation and comparison of mortality profiles in archaeozoology

Archaeozoological mortality profiles have been used to infer site-specific subsistence strategies. There is however no common agreement on the best way to present these profiles and confidence intervals around age class proportions. In order to deal with these issues, we propose the use of the Dirichlet distribution and present a new approach to perform age-at-death multivariate graphical comparisons. We demonstrate the efficiency of this approach using domestic sheep/goat dental remains from 10 Cardial sites (Early Neolithic) located in South France and the Iberian Peninsula. We show that the Dirichlet distribution in age-at-death analysis can be used: (i) to generate Bayesian credible intervals around each age class of a mortality profile, even when not all age classes are observed; and (ii) to create 95% kernel density contours around each age-at-death frequency distribution when multiple sites are compared using correspondence analysis. The statistical procedure we present is applicable to the analysis of any categorical count data and particularly well-suited to archaeological data (e.g. potsherds, arrow heads) where sample sizes are typically small.

Sound Art and Public Engagement in the Built Environment: Reflections from an Architecture Center

This article explores the deployment of sound in architectural-curatorial and community engagement contexts through the work of PLACE, a multidisciplinary not-for-profit architecture center in Belfast, Northern Ireland. The author, who worked with PLACE and contributed to the projects discussed here, contextualizes architecture centers and their relationship with sound before examining the specific case of sound and sound art in Northern Ireland and case studies of projects delivered by PLACE. Specifically, the article evaluates two sound installation artworks and three community engagement projects for young audiences. As a means of curating urbanism and architecture, sound-art-as-public-art affords useful strategies to examine, describe or critique the environment as alternatives to traditional architecture exhibition formats. Sound’s temporality and materiality allow sound art works to exist as temporary sculptural interventions in the urban sphere, with attendant implications for public art procurement and urban acoustics. Rich territories of engagement are opened when using sound in a community participatory context.

Modelling the effectiveness of grass buffer strips in managing muddy floods under a changing climate

Muddy floods occur when rainfall generates runoff on agricultural land, detaching and transporting sediment into the surrounding natural and built environment. In the Belgian Loess Belt, muddy floods occur regularly and lead to considerable economic costs associated with damage to property and infrastructure. Mitigation measures designed to manage the problem have been tested in a pilot area within Flanders and were found to be cost-effective within three years. This study assesses whether these mitigation measures will remain effective under a changing climate. To test this, the Water Erosion Prediction Project (WEPP) model was used to examine muddy flooding diagnostics (precipitation, runoff, soil loss and sediment yield) for a case study hillslope in Flanders where grass buffer strips are currently used as a mitigation measure. The model was run for present day conditions and then under 33 future site-specific climate scenarios. These future scenarios were generated from three earth system models driven by four representative concentration pathways and downscaled using quantile mapping and the weather generator CLIGEN. Results reveal that under the majority of future scenarios, muddy flooding diagnostics are projected to increase, mostly as a consequence of large scale precipitation events rather than mean changes. The magnitude of muddy flood events for a given return period is also generally projected to increase. These findings indicate that present day mitigation measures may have a reduced capacity to manage muddy flooding given the changes imposed by a warming climate with an enhanced hydrological cycle. Revisions to the design of existing mitigation measures within existing policy frameworks are considered the most effective way to account for the impacts of climate change in future mitigation planning. 

Leistungsverfügbarkeit und Echtzeitfähigkeit in Systemen intelligenter Ladungsträger

In diesem Artikel werden Leistungsverfügbarkeit und Echtzeitfähigkeit am Beispiel eines Lagersystems für intelligente Behälter betrachtet. Es wird dabei die Echtzeitfähigkeit dieses Systems anhand einer realistischen Fallstudie in einem Stetigfördersystem analysiert. Anhand dieser Analyse wird ein Konzept für die gezielte Planung eines Lagersystems für eine spezifische Leistungsverfügbarkeit entworfen. Kernbestandteil ist ein Kommunikationsprotokoll, das die Reservierung von Fördermitteln mittels logischer Zeit erlaubt. Über eine veränderte Reservierungsstrategie kann ein geeigneter Dispositionszeitpunkt berechnet werden, der eine Termineinhaltung aller Aufträge garantiert.

Ecological effects of flow and temperature regimes on fish communities in temperate rivers

Les organismes aquatiques sont adaptés à une grande variabilité hydrique et thermique des rivières. Malgré ceci, la régulation des eaux suscite des changements aux débits qui peuvent provoquer des impacts négatifs sur la biodiversité et les processus écologiques en rivière. Celle-ci peut aussi causer des modifications au niveau des régimes thermiques et des caractéristiques de l’habitat du poisson. Des données environnementales et biologiques décrivant l’habitat du poisson existent, mais elles sont incomplètes pour plusieurs rivières au Canada et de faible qualité, limitant les relations quantitatives débit-température-poissons à un petit nombre de rivières ou à une région étudiée. La recherche menée dans le cadre de mon doctorat concerne les impacts de la génération d'hydroélectricité sur les rivières; soit les changements aux régimes hydriques et thermiques reliés à la régulation des eaux sur la variation des communautés ichtyologiques qui habitent les rivières régulées et naturelles au Canada. Suite à une comparaison d’échantillonnage de pêche, une méthode constante pour obtenir des bons estimés de poisson (richesse, densité et biomasse des espèces) a été établie pour évaluer la structure de la communauté de poissons pour l’ensemble des rivières ciblées par l’étude. Afin de mieux comprendre ces changements environnementaux, les principales composantes décrivant ces régimes ont été identifiées et l’altération des régimes hydriques pour certaines rivières régulées a été quantifiée. Ces résultats ont servi à établir la relation significative entre le degré de changement biotique et le degré de changement hydrique pour illustrer les différences entre les régimes de régulation. Pour faire un complément aux indices biotiques déjà calculés pour l’ensemble des communautés de poissons (diversité, densité et biomasse des espèces par rivière), les différences au niveau des guildes de poissons ont été quantifiées pour expliquer les divers effets écologiques dus aux changements de régimes hydriques et thermiques provenant de la gestion des barrages. Ces derniers résultats servent à prédire pour quels traits écologiques ou groupes d’espèces de poissons les composantes hydriques et thermiques sont importantes. De plus, ces derniers résultats ont servi à mettre en valeur les variables décrivant les régimes thermiques qui ne sont pas toujours inclues dans les études hydro-écologiques. L’ensemble des résultats de cette thèse ont des retombées importantes sur la gestion des rivières en évaluant, de façon cohérente, l’impact de la régulation des rivières sur les communautés de poissons et en développant des outils de prévision pour la restauration des écosystèmes riverains.

What kind of expertise is needed for low energy construction

The construction industry is responsible for 40% of European Union (EU) end-use emissions but addressing this is problematic, as evident from the performance gap between design intention and on-site energy performance. There is a lack of the expertise needed for low energy construction (LEC) in the UK as the complex work processes involved require ‘energy literacy’ of all construction occupations, high qualification levels, broad occupational profiles, integrated teamworking, and good communication . This research identifies the obstacles to meeting these requirements, the nature of the expertise needed to break down occupational divisions and bridge those interfaces where the main heat losses occur, and the transition pathway implied. Obstacles include a decline in the level, breadth and quality of construction vocational education and training (VET), the lack of a learning infrastructure on sites, and a fragmented employment structure. To overcome these and develop enhanced understanding of LEC requires a transformation of the existing structure of VET provision and construction employment and a new curriculum based on a broader concept of agency and backed by rigorous enforcement of standards. This can be achieved through a radical transition pathway rather than market-based solutions to a low carbon future for the construction sector.

Macroinvertebrate Diversity in Urban and Rural Ponds: Implications for Freshwater Biodiversity Conservation

Ponds are among the most biodiverse freshwater ecosystems, yet face significant threats from removal, habitat degradation and a lack of legislative protection globally. Information regarding the habitat quality and biodiversity of ponds across a range of land uses is vital for the long term conservation and management of ecological resources. In this study we examine the biodiversity and conservation value of macroinvertebrates from 91 lowland ponds across 3 land use types (35 floodplain meadow, 15 arable and 41 urban ponds). A total of 224 macroinvertebrate taxa were recorded across all ponds, with urban ponds and floodplain ponds supporting a greater richness than arable ponds at the landscape scale. However, at the alpha scale, urban ponds supported lower faunal diversity (mean: 22 taxa) than floodplain (mean: 32 taxa) or arable ponds (mean: 30 taxa). Floodplain ponds were found to support taxonomically distinct communities compared to arable and urban ponds. A total of 13 macroinvertebrate taxa with a national conservation designation were recorded across the study area and 12 ponds (11 floodplain and 1 arable pond) supported assemblages of high or very high conservation value. Pond conservation currently relies on the designation of individual ponds based on very high biodiversity or the presence of taxa with specific conservation designations. However, this site specific approach fails to acknowledge the contribution of ponds to freshwater biodiversity at the landscape scale. Ponds are highly appropriate sites outside of protected areas (urban/arable), with which the general public are already familiar, for local and landscape scale conservation of freshwater habitats.

Type-Aware Programming Models for Distributed Applications

Thesis (Ph.D.)--University of Washington, 2016-08

Leave a light on – Terracotta lamps from Agia Paraskevi of Arachamitai, from the Classical period until Roman times

This thesis deals with terracotta lamps found at the archaeological site of Agia Paraskevi of Arachamitai during the years 2010–2014. The research questions are: What kind of lamps were used on the site and when? For what purposes were the lamps used? Where were they manufactured? In order to address these questions, a total of fifty lamps have been chosen to form the body of research material. To answer the first research question, a typology for these lamps has been created with twenty-one types and one variant, after examining various lamp typologies and previous publications. The find contexts and distribution of the lamps are both examined to test the dates for the different lamp types gathered from other publications, and to answer the question on what purposes the lamps were used on the site. Visual observation of the clay of the lamps and information on clay materials from other sites are both examined to determine the lamps' origins. The lamps in this study date from the 5th century BC to the 2nd century AD. The find contexts allow the dating of the lamps according to the other publications, but suggestions of more site-specific dates are given to some lamps. The distribution of the lamps indicates that while most were used for everyday lighting at the sanctuary, some may have had ritual connections. Most of the lamps were manufactured locally or at a nearby Arcadian site. Few lamps can be regarded as imports.