Molecular Mechanisms Regulating the Platelet Thrombin Receptor PAR4

Proteinase activated receptor 4 (PAR4) is a G-protein-coupled receptor with an important role in the platelet response to vascular injury. In platelets, PAR4 activation, by thrombin-cleavage of its N-terminus and unmasking of a tethered ligand, leads to G-protein- and beta-arrestin-mediated intracellular signalling pathways which result in platelet activation, shape change, and ultimately, platelet aggregation. As an important platelet thrombin receptor, PAR4 is an interesting target for the development of anti-platelet therapeutics. However, molecular determinants of PAR4 activation, signalling, and signal regulation remain poorly understood. In this thesis, mechanisms of PAR4 activation and signalling were studied through determination of the molecular basis of agonist binding at the extracellular surface of the receptor that result in G-protein signalling and beta-arrestin recruitment and evaluation of the role of Helix-8 and C-terminal tail residues on effector interaction and signalling. In our first series of experiments, we evaluated the impact of single amino acid substitutions to the parental PAR4 agonist peptide, AYPGKF-NH2, in an effort to understand the chemical characteristics enabling activity at the PAR4 receptor. We identified key residue characteristics contributing to agonist peptide activation of PAR4. We further showed that different chemical modifications affect G-protein and beta-arrestin signalling through PAR4, providing leads for further development of biased ligands of PAR4. Additionally, using in silico receptor modelling and peptide docking, we identify a key residue in the ECL2 of the receptor that is required for receptor activation by either peptide or enzyme-revealed tethered ligand. Subsequent studies evaluated the role of the Helix-8 and C-terminal tail motifs in PAR4 and the related receptor, PAR2, on intracellular effector interaction and signalling. PAR4 was compared to PAR2 since several key Class A GPCR regulatory sites are missing in PAR4 and are retained in PAR2. These studies have revealed residues and structural features that are involved in PAR4 interaction with G-proteins and beta-arrestin. Importantly, we also identified key differences in residues that are important for signalling following enzyme- versus peptide-activation of PARs. Taken together, this body of work enhances our understanding of how PAR4 engages and is activated by agonists to promote signalling and cellular function

Les vestiges humains gravettiens dans le Sud-Ouest de la France : bilan du projet Gravett’os

Cette communication présente les principaux résultats du projet Gravett’Os, qui porte sur du matériel anthropologique du Sud-Ouest de la France (découvertes récentes et reprises des collections anciennes) associé au Gravettien (34-24 000 cal BP). Ce projet a permis l’identification de 32 individus provenant de 5 sites (Cussac, Fournol, Gargas, Abri Pataud, Cro-Magnon). Nos études confortent les analyses précédentes sur les comportements au Gravettien : extrême mobilité et division sexuelle du..

The seeds of divergence: the economy of French North America, 1688 to 1760

Generally, Canada has been ignored in the literature on the colonial origins of divergence with most of the attention going to the United States. Late nineteenth century estimates of income per capita show that Canada was relatively poorer than the United States and that within Canada, the French and Catholic population of Quebec was considerably poorer. Was this gap long standing? Some evidence has been advanced for earlier periods, but it is quite limited and not well-suited for comparison with other societies. This thesis aims to contribute both to Canadian economic history and to comparative work on inequality across nations during the early modern period. With the use of novel prices and wages from Quebec—which was then the largest settlement in Canada and under French rule—a price index, a series of real wages and a measurement of Gross Domestic Product (GDP) are constructed. They are used to shed light both on the course of economic development until the French were defeated by the British in 1760 and on standards of living in that colony relative to the mother country, France, as well as the American colonies. The work is divided into three components. The first component relates to the construction of a price index. The absence of such an index has been a thorn in the side of Canadian historians as it has limited the ability of historians to obtain real values of wages, output and living standards. This index shows that prices did not follow any trend and remained at a stable level. However, there were episodes of wide swings—mostly due to wars and the monetary experiment of playing card money. The creation of this index lays the foundation of the next component. The second component constructs a standardized real wage series in the form of welfare ratios (a consumption basket divided by nominal wage rate multiplied by length of work year) to compare Canada with France, England and Colonial America. Two measures are derived. The first relies on a “bare bones” definition of consumption with a large share of land-intensive goods. This measure indicates that Canada was poorer than England and Colonial America and not appreciably richer than France. However, this measure overestimates the relative position of Canada to the Old World because of the strong presence of land-intensive goods. A second measure is created using a “respectable” definition of consumption in which the basket includes a larger share of manufactured goods and capital-intensive goods. This second basket better reflects differences in living standards since the abundance of land in Canada (and Colonial America) made it easy to achieve bare subsistence, but the scarcity of capital and skilled labor made the consumption of luxuries and manufactured goods (clothing, lighting, imported goods) highly expensive. With this measure, the advantage of New France over France evaporates and turns slightly negative. In comparison with Britain and Colonial America, the gap widens appreciably. This element is the most important for future research. By showing a reversal because of a shift to a different type of basket, it shows that Old World and New World comparisons are very sensitive to how we measure the cost of living. Furthermore, there are no sustained improvements in living standards over the period regardless of the measure used. Gaps in living standards observed later in the nineteenth century existed as far back as the seventeenth century. In a wider American perspective that includes the Spanish colonies, Canada fares better. The third component computes a new series for Gross Domestic Product (GDP). This is to avoid problems associated with using real wages in the form of welfare ratios which assume a constant labor supply. This assumption is hard to defend in the case of Colonial Canada as there were many signs of increasing industriousness during the eighteenth and nineteenth centuries. The GDP series suggest no long-run trend in living standards (from 1688 to circa 1765). The long peace era of 1713 to 1740 was marked by modest economic growth which offset a steady decline that had started in 1688, but by 1760 (as a result of constant warfare) living standards had sunk below their 1688 levels. These developments are accompanied by observations that suggest that other indicators of living standard declined. The flat-lining of incomes is accompanied by substantial increases in the amount of time worked, rising mortality and rising infant mortality. In addition, comparisons of incomes with the American colonies confirm the results obtained with wages— Canada was considerably poorer. At the end, a long conclusion is provides an exploratory discussion of why Canada would have diverged early on. In structural terms, it is argued that the French colony was plagued by the problem of a small population which prohibited the existence of scale effects. In combination with the fact that it was dispersed throughout the territory, the small population of New France limited the scope for specialization and economies of scale. However, this problem was in part created, and in part aggravated, by institutional factors like seigneurial tenure. The colonial origins of French America’s divergence from the rest of North America are thus partly institutional

Promoting Active Learning in Physiology Lectures Through Student Response Systems: To Click or Not to Click

Courses in physiology engage students through active learning strategies including small group discussions, group work, and opportunities to explore a scientific problem and explain their findings. Many of these active learning exercises take place in tutorial and laboratory settings. Unfortunately, traditional physiology lectures are often limited to conveying information through lecturing and PowerPoint slides. This approach provides little opportunity for student engagement above lower-order cognition, i.e., writing notes, listening, memorization (Freeman et al. 2014). Student response systems (e.g., clickers) are a valuable tool to facilitate active learning in the lecture setting that could enable students to take control of their learning (“Do I truly understand this topic/concept/theory?”) (Hwang, Wong, Lam & Lam 2015). In addition, clickers provide valuable instant feedback to the lecturer about student comprehension, and can be used to track participation and attendance. Many platforms are now available including clicker devices and virtual clickers to facilitate active learning and meta-cognitive exercises in the lecture setting. Student feedback response platforms may provide a way to introduce active learning into the lecture setting with physiology lectures resulting improved engagement and better achievement of learning outcomes. This workshop provides practical strategies and examples to help instructors evaluate the benefits, challenges, and methods of integrating student response systems into the physiology lecture setting

Biased signalling in platelet G-protein‐coupled receptors.

Platelets are small megakaryocyte-derived, anucleate, disk-like structures that play an outsized role in human health and disease. Both a decrease in the number of platelets and a variety of platelet function disorders result in petechiae or bleeding that can be life threatening. Conversely, the inappropriate activation of platelets, within diseased blood vessels, remains the leading cause of death and morbidity by affecting heart attacks and stroke. The fine balance of the platelet state in healthy individuals is controlled by a number of receptor-mediated signaling pathways that allow the platelet to rapidly respond and maintain haemostasis. G-protein coupled receptors (GPCRs) are particularly important regulators of platelet function. Here we focus on the major platelet-expressed GPCRs and discuss the roles of downstream signaling pathways (e.g., different G-protein subtypes or β-arrestin) in regulating the different phases of the platelet activation. Further, we consider the potential for selectively targeting signaling pathways that may contribute to platelet responses in disease through development of biased agonists. Such selective targeting of GPCR-mediated signaling pathways by drugs, often referred to as biased signaling, holds promise in delivering therapeutic interventions that do not present significant side effects, especially in finely balanced physiological systems such as platelet activation in haemostasis.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Relationships of climate and cell features in stems and roots of black spruce and balsam fir

• The anatomical differences of mature black spruces and balsam firs were examined at stem and root level in order to characterize their wood properties at cellular level and link these differences to climate. • Anatomical variability of these species was evaluated in relation to climate data gathered from 2001 to 2004 during the cell enlargement (CE) and wall thickening and lignification (WTL) phases. Lumen area, single cell wall thickness and total tracheid radial diameter were analyzed and regrouped into earlywood and latewood. • Results from a principal component analysis (PCA) indicated that both first eigenvectors account for 82% and 90% of total variance for CE and WTL respectively. These component factors revealed that precipitation, humidity and number of days with precipitation significantly influence the lumen area (p = 0.0168) and radial cell diameter (p = 0.0222) in earlywood. Significant differences were registered between species and tree parts (stem and root) for the lumen area, radial cell diameter and cell wall thickness in both earlywood and latewood. • In our study, black spruce exhibited smaller tracheid size in both stem and roots compared to balsam fir. Furthermore, the lower amount of tracheids produced during the growing season and higher proportion of latewood ensure a higher wood density of black spruce. The influence of temperature on earlywood formation is significant, whereas no influence was observed on latewood

High Affinity Fluorescent Probe for Proteinase-Activated Receptor 2 (PAR2)

© 2019 American Chemical Society. PAR2 is a proteolytically activated G protein-coupled receptor (GPCR) that is implicated in various cancers and inflammatory diseases. Ligands with low nanomolar affinity for PAR2 have been developed, but there is a paucity of research on the development of PAR2-targeting imaging probes. Here, we report the development of seven novel PAR2-targeting compounds. Four of these compounds are highly potent and selective PAR2-targeting peptides (EC50 = 10 to 23 nM) that have a primary amine handle available for facile conjugation to various imaging components. We describe a peptide of the sequence Isox-Cha-Chg-ARK(Sulfo-Cy5)-NH2 as the most potent and highest affinity PAR2-selective fluorescent probe reported to date (EC50 = 16 nM, KD = 38 nM). This compound has a greater than 10-fold increase in potency and binding affinity for PAR2 compared to the leading previously reported probe and is conjugated to a red-shifted fluorophore, enabling in vitro and in vivo studies