The design and implementation of a peer learning programme in physics utilising undergraduate peer leaders: an in-depth investigation into the effect of participation and the experience of the peer leaders

Physics Education Research (PER) has shown that students leave the physics classroom holding the same misconceptions about physics they had when they entered. PER has demonstrated that these misconceptions are often deeply held and are difficult to change. The literature indicates that Peer Learning has advantages compared with traditional methods in producing conceptual change and that there are a number of potential benefits for utilising undergraduates as Peer Leaders in Peer Learning programmes. Gartner, Kohler, and Riessman (1971) summarised a number of beneficial cognitive processes that occur when Peer Leaders prepare for Peer Learning sessions. It is believed that in reviewing, organising and teaching the material, they may gain a better understanding of the subject. However, little or no literature exists on the effect participation has on Peer Leaders or their experience of a Peer Learning programme. In particular, few studies determining the effect of participation on the Peer Leaders in physics at third level exist. The purpose of this study is to explore the effect of becoming a Peer Leader, to investigate the experience of becoming a Peer Leader, and to illuminate the implications of using undergraduate Peer Leaders in physics tutorials at third level. A model of Peer Learning was designed and implemented in physics tutorials at the University of Limerick and the Peer Leaders selected for this study had direct experience of undergraduate introductory physics courses. This Peer Learning model involved students working in cooperative groups with an undergraduate Peer Leader as a facilitator of their learning. Training was provided to the Peer Leaders to ensure that they created an environment where the students were actively and productively engaged with the material and with each other. Action research was chosen as the central methodology of this thesis, within this, two cases and several mixed research methods were employed. Conceptual tests, questionnaires, semi-structured interviews and application forms were the methods of data collection. The action cycles of the study contained two different cases of Peer Leaders recruited from the third year classes of students following concurrent science teacher education programmes at the University of Limerick. This thesis presents findings on the effects of participation on the Peer Leaders, their experience of the programme, and the implications for utilising undergraduate Peer Leaders in physics tutorials. Peer Leaders responses on the change in their conceptual understanding are reported along with the benefits that they claim to have experienced. Findings on the Peer Leaders misconceptions in Introductory Physics and their views on how they learn physics as a student and as a teacher are presented

Laminin-411 Is a Vascular Ligand for MCAM and Facilitates TH17 Cell Entry into the CNS

<div><p>TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 <em>in vitro</em>. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.</p> </div

Label Free Fragment Screening Using Surface Plasmon Resonance as a Tool for Fragment Finding – Analyzing Parkin, a Difficult CNS Target

<div><p>Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.</p></div

Functionally active FL-Parkin binds three different protein ligands.

<p>FL-FLAG Parkin was captured on a CM5 sensor chip with immobilized anti-FLAG antibody at a stoichiometry of (3∶1) (Parkin:Ab). Each of the protein ligands was injected at concentrations above 10-fold K_D if possible: (a) Ubiquitin at 500, 250, 125, 62.5, 31.25 and 15.62 μM (b) His-UblD and (c) UbcH7 were injected at 140, 46.7, 15.6, 5.2, 1.7 and 0.6 μM over FL-FLAG Parkin. All data was fitted to 1∶1 binding model. Each binding test was repeated at different test occasions (n≥3).1∶1 binding isotherms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s004" target="_blank">Figure S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s006" target="_blank">6</a>.</p

Affinities K_D (μM) of protein ligands to different Parkin proteins.

1<p><b>Ubiquitin-like domain of Parkin; ²Ubiquitin conjugating enzyme E2; ³Ubiquitin; ⁴ Full-length.</b></p

Thermal treated FL-FLAG-Parkin is more active than non-thermal treated FL-FLAG-Parkin independent of reducing agent.

<p>TR-FRET S5a assay using 150 nM FL-FLAG Parkin and 200nM biotinylated S5a substrate in the presence or absence of 5 mM reducing agent. FL-FLAG Parkin was incubated at 56°C for 30 min and then cooled to RT (thermal treated). Thermal treated FL-FLAG Parkin exhibit significantly different levels of Parkin activity in presence of each of the three reducing agents p<0.0001 (hatched bar) (n = 4). Non thermal treated FL-FLAG Parkin showed similar levels of activity in presence of either DTT or TCEP with p = 0.5368 and both activities are significantly higher than in presence of BME with p<0.0001 (white bar) (n = 4).</p

hMCAM binds to a ligand in the ECM with identical staining to laminin α4.

<p>Calcein labeled, hMCAM expressing MOLT 4 cells were preincubated with either isotype control (A) or anti-hMCAM (clone 17) (B) followed by incubation on tissue sections from healthy mice. After gentle washing of unbound cells, and mounting with DAPI, bound cells were visualized by fluorescent microscopy. Healthy mouse brain sections containing choroid plexus were stained with fluorescently labeled mMCAM-Fc protein and pan-laminin antibody. Staining of mMCAM-Fc was detected on choroid plexus (C) as well as the vasculature throughout the tissues (D). Fluorescently labeled mMCAM-Fc protein was preincubated with either isotype control (E) or anti-mMCAM (clone 15) (F) before addition to tissue sections of healthy mouse brain. Healthy mouse tissues were stained with fluorescently labeled mMCAM-Fc and CD31 (G) anti-mMCAM and pan laminin (H) or anti-mMCAM alone (I). Tissues from mice with active EAE were stained with fluorescently labeled mMCAM-Fc and pan-laminin (J) or mMCAM-Fc and an antibody specific to the α4 chain of laminin (K).</p

Ligand efficiencies of confirmed SPR hits.

<p>Plot of ligand efficiencies (LE) of SPR confirmed hits vs. number of heavy atoms <i>N_h.</i> Binding affinities (K_D) were obtained from injection of fragments in dose response manner from 50 uM in two-fold dilutions. One very interesting hit with LE of 0.5 and <i>HAC</i> of 13 is A (red dot) with a mid micro-molar affinity (K_D of 18.6 uM).</p

Physico-chemical properties of SPR hits vs. fragment library.

<p>The distribution of compounds in the fragment library is shown as circles; the SPR hits as triangles. The distribution of SPR hits is consistent with the fragment library (though noisier because of the small number of compounds), with the exception of hydrogen bond donors, which are over-represented in the SPR hits compared to fragment library.</p

Dithiothreitol (DTT) binds to non-thermal treated FL-FLAG Parkin.

<p>(A) SPR data: DTT was injected at 62.5, 31.25, 15.62, 7.8, 3.9, 1.95 and 0.98, 1.9 μM in 50 mM HEPES pH 8.8, 0.005% Tween-20, 0.01% PF-127. The kinetic fits are shown in red. The affinity was determined to 1.4 μM at 47% Rmax (Rmax: 34 RU). DTT dissociates very slowly and the sensorgrams show ill-behavior above 16.5 μM. SPR data for thermal treated FL-FLAG Parkin was similar (data not shown) (B) NMR-STD: (a) Proton spectrum of 0.25 mM compound Z (black); (b) STD spectrum of 0.25 mM compound Z (green); (c) STD spectrum of 0.25 mM compound Z in the presence of 4 µM FL Parkin protein (blue); (d) STD spectrum of 0.25 mM compound Z in the presence of 4 µM FL Parkin protein and 0.5 mM DTT (red).</p