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

Genetic and Molecular Basis of Encapsulation and Capsule Diversity in Kingella kingae

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

Standing stocks and carbon isotopes of live benthic foraminifera from the South Atlantic

Live (Rose Bengal stained) and dead benthic foraminifera of surface and subsurface sediments from 25 stations in the eastern South Atlantic Ocean and the Atlantic sector of the Southern Ocean were analyzed to decipher a potential influence of seasonally and spatially varying high primary productivity on the stable carbon isotopic composition of foraminiferal tests. Therefore, stations were chosen so that productivity strongly varied, whereas conservative water mass properties changed only little. To define the stable carbon isotopic composition of dissolved inorganic carbon (d13CDIC) in ambient water masses, we compiled new and previously published d13CDIC data in a section running from Antarctica through Agulhas, Cape and Angola Basins, via the Guinea Abyssal Plain to the Equator. We found that intraspecific d13C variability of all species at a single site is constantly low throughout their distribution within the sediments, i.e. species specific and site dependent mean values calculated from all subbottom depths on average only varied by +/-0.09 per mil. This is important because it makes the stable carbon isotopic signal of species independent of the particular microhabitat of each single specimen measured and thus more constant and reliable than has been previously assumed. So-called vital and/or microhabitat effects were further quantified: (1) d13C values of endobenthic Globobulimina affinis, Fursenkoina mexicana, and Bulimina mexicana consistently are by between -1.5 and -1.0 per mil VPDB more depleted than d13C values of preferentially epibenthic Fontbotia wuellerstorfi, Cibicidoides pachyderma, and Lobatula lobatula. (2) In contrast to the Antarctic Polar Front region, at all stations except one on the African continental slope Fontbotia wuellerstorfi records bottom water d13CDIC values without significant offset, whereas L. lobatula and C. pachyderma values deviate from bottom water values by about -0.4 per mil and -0.6 per mil, respectively. This adds to the growing amount of data on contrasting cibicid d13C values which on the one hand support the original 1:1-calibration of F. wuellerstorfi and bottom water d13CDIC, and on the other hand document severe depletions of taxonomically close relatives such as L. lobatula and C. pachyderma. At one station close to Bouvet Island at the western rim of Agulhas Basin, we interpret the offset of -1.5 per mil between bottom water d13CDIC and d13C values of infaunal living Bulimina aculeata in contrast to about -0.6 +/- 0.1 per mil measured at eight stations close-by, as a direct reflection of locally increased organic matter fluxes and sedimentation rates. Alternatively, we speculate that methane locally released from gas vents and related to hydrothermal venting at the mid-ocean ridge might have caused this strong depletion of 13C in the benthic foraminiferal carbon isotopic composition. Along the African continental margin, offsets between deep infaunal Globobulimina affinis and epibenthic Fontbotia wuellerstorfi as well as between shallow infaunal Uvigerina peregrina and F. wuellerstorfi, d13C values tend to increase with generally increasing organic matter decomposition rates. Although clearly more data are needed, these offsets between species might be used for quantification of biogeochemical paleogradients within the sediment and thus paleocarbon flux estimates. Furthermore, our data suggest that in high-productivity areas where sedimentary carbonate contents are lower than 15 weight %, epibenthic and endobenthic foraminiferal d13C values are strongly influenced by 13C enrichment probably due to carbonate-ion undersaturation, whereas above this sedimentary carbonate threshold endobenthic d13C values reflect depleted pore water d13CDIC values.

(Table S1) TEX86 and BIT record from sediment core PBBC-1

A molecular organic geochemical proxy (TEX86) for sea surface temperature (SST) is compared with a foraminifera-based SST proxy (Mg/Ca) in a decadal-resolution marine sedimentary record spanning the last 1000 years from the Gulf of Mexico. We assess the relative strengths of the organic and inorganic paleoceanographic techniques for reconstructing high-resolution SST variability during recent climate events, including the Little Ice Age (LIA) and the Medieval Warm Period (MWP). SST estimates based on the molecular organic proxy TEX86 show a similar magnitude and pattern of SST variability to foraminiferal Mg/Ca-SST estimates but with some important differences. For instance, both proxies show a cooling (1°C-2°C) of Gulf of Mexico SSTs during the LIA. During the MWP, however, Mg/Ca-SSTs are similar to near-modern SSTs, while TEX86 indicates SSTs that were cooler than modern. Using the respective SST calibrations for each proxy results in TEX86-SST estimates that are 2°C-4°C warmer than Mg/Ca-SST throughout the 1000 year record. We interpret the TEX86-SST as a summer-weighted SST signal from the upper mixed layer, whereas the Mg/Ca-SST better reflects the mean annual SST. Downcore differences in the SST estimates between the two proxies (DeltaT = TEX86 - Mg/Ca) are interpreted in the context of varying seasonality and/or changing water column temperature gradients.

(Table 1) Stable oxygen isotope ratios of interstitial waters from ODP Sites 164-994 and 164-997

The d18O values of interstitial waters from Site 994 and Site 997 sediments, Blake Ridge, western Atlantic, tend to decrease with depth from 0.3 per mil to -0.5 per mil Standard Mean Ocean Water in the upper 200 mbsf, then fluctuate with significant positive spikes of Delta = 0.2 per mil - 0.5 per mil in the gas hydrate zone (200 to 450 mbsf), and finally increase from -0.4 per mil to -0.2 per mil toward 700 mbsf. Positive shifts of d18O IW in the gas hydrate zone are probably caused by the dissociation of gas hydrates originally contained in sediment cores. Gas hydrates recovered from the sites are enriched in 18O, d18O ranging between 2.7 per mil and 3.5 per mil. d18O values of gas hydrates and ambient interstitial waters give an oxygen isotopic fractionation factor of 1.0034-1.0040 at 12°-16°C and ~31 MPa (3 km below sea level). Based on this fractionation and observed isotopic anomalies in the gas hydrate zone, gas hydrates occupy 6% to 12% of pore-space volume within Blake Ridge sediments.

Oligocene to early Miocene benthic foraminifera in the eastern Equatorial Pacific

We investigated Oligocene and early Miocene benthic foraminiferal faunas (> 105 µm in size) from Ocean Drilling Program (Leg 199) Site 1218 (4826 m water depth and ~3300 to ~4000 m paleo-water depth) and Site 1219 (5063 m water depth and ~4200 to ~4400 m paleo-water depth) to understand the response of abyssal benthic foraminifera to mid-Oligocene glacial events in the eastern Equatorial Pacific Ocean. Two principal factor assemblages were recognized. The Factor 1 assemblage (common Nuttallides umbonifer) is related to either an influx of the Southern Component Water (SCW), possibly carbonate undersaturated, or a decrease in seasonality of the food supply from the surface ocean. The Factor 2 assemblage is characterized by typical deep-sea taxa living under variable trophic conditions, possibly with a seasonal component in food supply. The occurrence of abyssal benthic foraminifera faunas during the mid-Oligocene depends on either the effect of SCW or the seasonality of food resources. The Factor 1 assemblage was most common near 76Ol-C11r, 73Ol-C10rn and 67Ol-C9n (ca. 30.2, 29.1 and 26.8 Ma respectively by Pälike et al. (2006, doi:10.1126/science.1133822)). This indicates that the effect of SCW increased or the seasonal input of food from the surface ocean to benthic environments was weakened close to these glacial events. In contrast, the huge export flux of small biogenic carbonate particles close to these glacial events might be responsible for carbonate-rich sediments buffering carbonate undersaturation. Changes in deep-water masses or the periodicity of food supply from the surface ocean and variation in surface carbonate production affected by orbital forcing had an impact on the mid-Oligocene faunas of abyssal benthic foraminifera around the intervals of glacial events in the eastern Equatorial Pacific Ocean. The Factor 1 assemblage decreased sharply at ? 30 Ma (29.8 Ma by Pälike et al. (2006), 30.0 Ma by CK95) and returned to dominance after ? 29 Ma (28.6 Ma by Pälike et al. (2006), 28.8 Ma by CK95). It is likely that the effect of SCW (possibly carbonate undersaturated) has intensified since the late Oligocene. The faunal transition of benthic foraminifera in the eastern Equatorial Pacific Ocean at ~29 Ma might be attributable to the influence of Northern Component Water (NCW) input to the Southern Ocean and the subsequent formation of SCW at about that time.

EUSAAR size distribution analysis database

Two years of harmonized aerosol number size distribution data from 24 European field monitoring sites have been analysed. The results give a comprehensive overview of the European near surface aerosol particle number concentrations and number size distributions between 30 and 500 nm of dry particle diameter. Spatial and temporal distribution of aerosols in the particle sizes most important for climate applications are presented. We also analyse the annual, weekly and diurnal cycles of the aerosol number concentrations, provide log-normal fitting parameters for median number size distributions, and give guidance notes for data users. Emphasis is placed on the usability of results within the aerosol modelling community. We also show that the aerosol number concentrations of Aitken and accumulation mode particles (with 100 nm dry diameter as a cut-off between modes) are related, although there is significant variation in the ratios of the modal number concentrations. Different aerosol and station types are distinguished from this data and this methodology has potential for further categorization of stations aerosol number size distribution types. The European submicron aerosol was divided into characteristic types: Central European aerosol, characterized by single mode median size distributions, unimodal number concentration histograms and low variability in CCN-sized aerosol number concentrations; Nordic aerosol with low number concentrations, although showing pronounced seasonal variation of especially Aitken mode particles; Mountain sites (altitude over 1000 m a.s.l.) with a strong seasonal cycle in aerosol number concentrations, high variability, and very low median number concentrations. Southern and Western European regions had fewer stations, which decreases the regional coverage of these results. Aerosol number concentrations over the Britain and Ireland had very high variance and there are indications of mixed air masses from several source regions; the Mediterranean aerosol exhibit high seasonality, and a strong accumulation mode in the summer. The greatest concentrations were observed at the Ispra station in Northern Italy with high accumulation mode number concentrations in the winter. The aerosol number concentrations at the Arctic station Zeppelin in Ny-Ålesund in Svalbard have also a strong seasonal cycle, with greater concentrations of accumulation mode particles in winter, and dominating summer Aitken mode indicating more recently formed particles. Observed particles did not show any statistically significant regional work-week or weekday related variation in number concentrations studied. Analysis products are made for open-access to the research community, available in a freely accessible internet site. The results give to the modelling community a reliable, easy-to-use and freely available comparison dataset of aerosol size distributions.

Concentrations and abundance ratios of long-chain alkenones and glycerol dialkyl glycerol tetraethers in sinking particles south of Java

In this study, we obtained concentrations and abundance ratios of long-chain alkenones and glycerol dialkyl glycerol tetraethers (GDGTs) in a one-year time-series of sinking particles collected with a sediment trap moored from December 2001 to November 2002 at 2200 m water depth south of Java in the eastern Indian Ocean. We investigate the seasonality of alkenone and GDGT fluxes as well as the potential habitat depth of the Thaumarchaeota producing the GDGTs entrained in sinking particles. The alkenone flux shows a pronounced seasonality and ranges from 1 µg m-**2 d**-1 to 35 µg m**-2 d**-1. The highest alkenone flux is observed in late September during the Southeast monsoon, coincident with high total organic carbon fluxes as well as high net primary productivity. Flux-weighted mean temperature for the high flux period using the alkenone-based sea-surface temperature (SST) index UK'37 is 26.7°C, which is similar to satellite-derived Southeast (SE) monsoon SST (26.4°C). The GDGT flux displays a weaker seasonality than that of the alkenones. It is elevated during the SE monsoon period compared to the Northwest (NW) monsoon and intermonsoon periods (approximately 2.5 times), which is probably related to seasonal variation of the abundance of Thaumarchaeota, or to enhanced export of GDGTs by aggregation with sinking phytoplankton detritus. Flux-weighted mean temperature inferred from the GDGT-based TEXH86 index is 26.2°C, which is 1.8 °C lower than mean annual (ma) SST but similar to SE monsoon SST. As the time series of TEXH86 temperature estimates, however, does not record a strong seasonal amplitude, we infer that TEXH86 reflects ma upper thermocline temperature at approximately 50 m water depth.

Sea-surface reconstructions of the western Pacific Ocean during the last 5.3 million years

The late Neogene was a time of cryosphere development in the northern hemisphere. The present study was carried out to estimate the sea surface temperature (SST) change during this period based on the quantitative planktonic foraminiferal data of 8 DSDP sites in the western Pacific. Target factor analysis has been applied to the conventional transfer function approach to overcome the no-analog conditions caused by evolutionary faunal changes. By applying this technique through a combination of time-slice and time-series studies, the SST history of the last 5.3 Ma has been reconstructed for the low latitude western Pacific. Although the present data set is close to the statistical limits of factor analysis, the clear presence of sensible variations in individual SST time-series suggests the feasibility and reliability of this method in paleoceanographic studies. The estimated SST curves display the general trend of the temperature fluctuations and reveal three major cool periods in the late Neogene, i.e. the early Pliocene (4.7 3.5 Ma), the late Pliocene (3.1-2.7 Ma), and the latest Pliocene to early Pleistocene (2.2-1.0 Ma). Cool events are reflected in the increase of seasonality and meridional SST gradient in the subtropical area. The latest Pliocene to early Pleistocene cooling is most important in the late Neogene climatic evolution. It differs from the previous cool events in its irreversible, steplike change in SST, which established the glacial climate characteristic of the late Pleistocene. The winter and summer SST decreased by 3.3-5.4°C and 1.0 2.1C in the subtropics, by 0.9°C and 0.6C in the equatorial region, and showed little or no cooling in the tropics. Moreover, this cooling event occurred as a gradual SST decrease during 2.2 1.0 Ma at the warmer subtropical sites, while that at cooler subtropical site was an abrupt SST drop at 2.2 Ma. In contrast, equatorial and tropical western Pacific experienced only minor SST change in the entire late Neogene. In general, subtropics was much more sensitive to climatic forcing than tropics and the cooling events were most extensive in the cooler subtropics. The early Pliocene cool periods can be correlated to the Antarctic ice volume fluctuation, and the latest Pliocene early Pleistocene cooling reflects the climatic evolution during the cryosphere development of the northern hemisphere.

Academic Integrity and Senior Nursing Undergraduate Clinical Practice

Background: Academic integrity (AI) has been defined as the commitment to the values of honesty, trust, fairness, respect, and responsibility with courage in all academic endeavours. The senior years of nursing studies provide an intersection for students to transition to professional roles through student clinical practice. It is essential to understand what predicts senior nursing students’ intention to behave with AI so that efforts can be directed to initiatives focused on strengthening their commitment to behaving with AI. Research Questions: To what extent do students differ on Theory of Planned Behaviour (TPB) variables? What predicts intention to behave with academic integrity among senior nursing students in clinical practice across three different Canadian Schools of Nursing? Method: The TPB framework, an elicitation (n=30) and two pilot studies (n=59, n=29) resulted in the development of a 38 question (41-item) self-report survey (Miron Academic Integrity Nursing Survey—MAINS: α>0.70) that was administered to Year 3 and 4 students (N=339). Three predictor variables (attitude, subjective norm, perceived behavioural control) were measured with students’ intention to behave with AI in clinical. Age, sex, year of study, program stream, students’ understanding of AI policies, and locations where students accessed AI information were also measured. Results: Hierarchical multiple regression analyses revealed that background, site, and TPB variables explained 32.6% of the variance in intention to behave with academic integrity. The TPB variables explained 26.8% of the variance in intention after controlling for background and site variables. In the final model, only the TPB predictor variables were statistically significant with Attitude having the highest beta value (beta=0.35, p<0.001), followed by Subjective Norm (beta=0.21, p<0.001) and Perceived Behavioural Control (beta=0.12, p<0.02). Conclusion: Student attitude is the strongest predictor to intention to behave with AI in clinical practice and efforts to positively influence students’ attitudes need to be a focus for schools, curricula, and clinical educators. Opportunities for future research should include replicating the current study with students enrolled in other professional programs and intervention studies that examine the effectiveness of specific endeavours to promote AI in practice.

Effects of retirement on cognitive function: A gender and international perspecive

Background: As the global population is ageing, studying cognitive impairments including dementia, one of the leading causes of disability in old age worldwide, is of fundamental importance to public health. As a major transition in older age, a focus on the complex impacts of the duration, timing, and voluntariness of retirement on health is important for policy changes in the future. Longer retirement periods, as well as leaving the workforce early, have been associated with poorer health, including reduced cognitive functioning. These associations are hypothesized to differ based on gender, as well as on pre-retirement educational and occupational experiences, and on post-retirement social factors and health conditions. Methods: A cross-sectional study is conducted to determine the relationship between duration and timing of retirement and cognitive function, using data from the five sites of International Mobility in Aging Study (IMIAS). Cognitive function is assessed using the Leganes Cognitive Test (LCT) scores in 2012. Data are analyzed using multiple linear regressions. Analyses are also done by site/region separately (Canada, Latin America, and Albania). Robustness checks are done with an analysis of cognitive change from 2012 to 2014, the effect of voluntariness of retirement on cognitive function. An instrumental variable (IV) approach is also applied to the cross-sectional and longitudinal analyses as a robustness check to address the potential endogeneity of the retirement variable. Results: Descriptive statistics highlight differences between men and women, as well as between sites. In linear regression analysis, there was no relationship between timing or duration of retirement and cognitive function in 2012, when adjusting for site/region. There was no association between retirement characteristics and cognitive function in site/region/stratified analyses. In IV analysis, longer retirement and on time or late retirement was associated with lower cognitive function among men. In IV analysis, there is no relationship between retirement characteristics and cognitive function among women. Conclusions: While results of the thesis suggest a negative effect of retirement on cognitive function, especially among men, the relationship remains uncertain. A lack of power results in the inability to draw conclusions for site/region-specific analysis and site-adjusted analysis in both linear and IV regressions.

(MIS)PERCEIVED DRINKING NORMS AND HAZARDOUS DRINKING BEHAVIOURS IN UNIVERSITY FIRST-YEAR UNDERGRADUATE STUDENTS AND THE EFFECTS OF GENDER

Purpose: Across Canada, undergraduate university students are one of the highest alcohol-consuming populations. Many students engage in hazardous drinking and are at risk for negative health and social consequences. Social Norms Theory suggests that students’ overestimation of drinking norms can result in an increase in their drinking behaviour. As of yet, none of the literature addresses the possible link between drinking norm (mis)perception and hazardous drinking in a Canadian undergraduate context. This is the first Canadian study to examine this potential association in first-year undergraduate students across multiple universities using gender as an effect modifier. Methods: Using data collected by the Caring Campus Project, for 2347 first-year students from three Canadian universities, I evaluated the prevalence of drinking norm misperceptions by site and gender. Using multiple-logistic regression models, I analyzed the relationship between misperceived drinking norms and hazardous drinking behaviours (assessed via AUDIT-C). Results: The proportion of students who overestimated drinking and binge drinking frequency norms varied by site and gender. There was a positive relationship between overestimated drinking/ binge drinking frequency norms and hazardous drinking, modified by gender. Controlling for living arrangement and site, the odds of female students being hazardous drinkers increased by a factor of 2.27 (CI: 1.73-2.99) when the drinking frequency norm was overestimated. A non-significant association was found for male students. Among female students, when living arrangement and site were controlled, the odds of being a hazardous drinker were 1.83 (0.84-3.95) and 2.69 (1.24-5.83) times greater when the drinking frequency norm was perceived at “2-4 times per month” and “2 or more times per week”, respectively. Among male students, when living arrangement, previous residence and site were controlled, the odds of being a hazardous drinker were 4.03 (2.62-6.19) and 8.54 (5.41-13.49) times greater when the binge drinking frequency norm was perceived at “2-4 times per month” and “2 or more times per week”, respectively. Conclusion: This novel study enhances the understanding of the association between (mis)perceived drinking norms and drinking behaviours in Canadian undergraduate students. The demonstrated importance of gender and site provides a strong impetus for Canadian universities to develop targeted alcohol reduction interventions.

Site Machines

Thesis (Ph.D.)--University of Washington, 2016-07

Macroinvertebrate communities and habitat structure in least-impacted hill streams of central Portugal

We surveyed macroinvertebrate communities in 31 hill streams in the Vouga River and Mondego River catchments in central Portugal. Despite applying a "least-impacted" criterion, channel and bank management was common, with 38% of streams demonstrating channel modification (damming) and 80% with evidence of bank modification. Principal component analysis (PCA) at the family and species level related the macroinvertebrates to habitat variables derived at three spatial scales -- site (20 m), reach (200 m), and catchment. Variation in community structure between sites was similar at the species and family level and was statistically related to pH, conductivity, temperature, flow, shade, and substrate size at the site scale; channel and bank habitat and riparian vegetation and land-use at the reach scale; and altitude and slope at the catchment scale. While the effects of river management were apparent in various ecologically important habitat features at the site and reach scale, a direct relationship with macroinvertebrate assemblages was only apparent between the extent of walled banks and the secondary PCA axis described by species data. The strong relationship between catchment scale variables and descriptors of physical structure at the reach and site scale suggests that catchment-scale parameters are valuable predicators of macroinvertebrate community structure in these streams despite the anthropogenic modifications of the natural habitat.

Phase I Environmental Site Assessment of the Proposed State Highway 60 Bypass Around Le Mars, Iowa, Plymouth County, Abridged Phase I, 2001

This report serves as a record of the Assessment activities, which consisted of a review of local, county, state, and federal records, interviews, and site reconnaissance. Sections 2 through 4 of this report document information gathered. during the records search and site visits for the Assessment. Section 5 presents the findings and conclusions of the Assessment.