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41	Étudier les fonctions des protéines avec des nanoantennes fluorescentes Harroun, Scott G. 09 1900 (has links) Caractériser la fonction des protéines est crucial pour notre compréhension des mécanismes moléculaires de la vie, des maladies, et aussi pour inspirer de nouvelles applications en bionanotechnologie. Pour y arriver, il est nécessaire de caractériser la structure et la dynamique de chaque état occupé par la protéine durant sa fonction. La caractérisation expérimentale des états transitoires des protéines représente encore un défi majeur parce que les techniques à haute résolution structurelle, telles que la spectroscopie RMN et la cristallographie aux rayons X, peuvent difficilement être appliquées à l’étude des états de courte durée. De plus, les techniques à haute résolution temporelle, telles que la spectroscopie de fluorescence, nécessitent généralement une chimie complexe pour introduire des fluorophores à des endroits spécifiques dans la protéine. Dans cette thèse nous introduisons l’utilisation des nanoantennes fluorescentes en tant que nouvelle stratégie pour détecter et signaler les changements de conformation des protéines via des interactions non covalentes entre des fluorophores spécifiques et la surface de la protéine. En utilisant des expériences et des simulations moléculaires, nous démontrons que des fluorophores chimiquement divers peuvent se lier et être utilisés pour sonder différentes régions d’une enzyme modèle, la phosphatase alcaline (PA). Ces nanoantennes peuvent être fixées directement aux protéines ou utilisées à l'aide du système de fixation simple et modulaire, le complexe biotine-streptavidine (SA), qui permet un criblage rapide et efficace de la nanoantenne optimale tant dans sa composition que sa longueur. Dans le cas de la PA, nous montrons que nos nanoantennes permettent la détection et la caractérisation des conformations distinctes incluant les changements conformationnels nanoscopiques produisant durant la catalyse du substrat. Nous démontrons également que les signaux fluorescents émis par la nanoantenne peuvent également permettre de caractériser la cinétique enzymatique d’une protéine en une seule expérience tout en incluant la détermination des paramètres « Michaelis-Menten » de ses substrats et inhibiteurs. Nous avons également exploré l'universalité de la stratégie ces nanoantennes fluorescentes en utilisant une autre protéine modèle, la Protéine G et son interaction avec les anticorps, et avons démontré son utilité pour mettre au point un essai permettant de détecter les anticorps. Ces nanoantennes simples et faciles à utiliser peuvent être appliquées pour détecter et analyser les changements conformationnels de toutes tailles et nos résultats suggèrent qu'elles pourraient être utilisées pour caractériser n’importe quel type de fonction. / The characterisation of protein function is crucial to understanding the molecular mechanisms of life and disease, and inspires new applications in bionanotechnology. To do so, it is necessary to characterise the structure and dynamics of each state that proteins adopt during their function. Experimental study of protein transient states, however, remains a major challenge because high-structural-resolution techniques, including NMR spectroscopy and X-ray crystallography, can often not be directly applied to study short-lived protein states. On the other hand, high-temporal-resolution techniques, such as fluorescence spectroscopy, typically require complicated site-specific labelling chemistry. This thesis introduces the use of fluorescent nanoantennas as a new strategy for sensing and reporting on protein conformational changes through noncovalent dye-protein interactions driven by a high local concentration. Using experiments and molecular simulations, we first demonstrate that chemically diverse dyes can bind and be used to probe different regions of a model enzyme, intestinal alkaline phosphatase (AP). These nanoantennas can be attached directly to proteins or employed using the simple and modular biotin-streptavidin (SA) attachment system, which enables rapid and efficient screening for high sensitivity by tuning their length and composition. We show that these nanoantennas enable the detection and characterisation of distinct conformational changes of AP, including nanoscale conformational changes that occur during substrate catalysis. We also show that the fluorescent signal emitted by the nanoantenna enables complete characterisation of enzyme kinetics in one experiment, including determination of Michaelis-Menten parameters of substrates and inhibitors of AP. We then explored the universality of the nanoantenna strategy by using a different model protein system. Protein G was shown to interact with antibodies, using a rapid screening strategy for antibody detection. These effective and easy-to-use nanoantennas could potentially be employed to monitor various conformational changes, and our results offer potential for characterising various protein functions. fonction des protéines cinétique des enzymes interaction protéine-protéine nanotechnologie de l’ADN nanoantenne spectroscopie fluorescence phosphatase alcaline biotine-streptavidine Protéine G inhibiteur enzymatique biosenseur protein function enzyme kinetics protein-protein interaction DNA nanotechnology nanoantenna fluorescence spectroscopy alkaline phosphatase biotin-streptavidin Protein G enzyme inhibitor biosensor
42	Evaluation of a Novel Biochemistry Course-Based Undergraduate Research Experience (CURE) Stefan M Irby (6326255) 15 May 2019 (has links) <p>Course-based Undergraduate Research Experiences (CUREs) have been described in a range of educational contexts. Although various learning objectives, termed anticipated learning outcomes (ALOs) in this project, have been proposed, processes for identifying them may not be rigorous or well-documented, which can lead to inappropriate assessment and speculation about what students actually learn from CUREs. Additionally, evaluation of CUREs has primarily relied on student and instructor perception data rather than more reliable measures of learning.This dissertation investigated a novel biochemistry laboratory curriculum for a Course-based Undergraduate Research Experience (CURE) known as the Biochemistry Authentic Scientific Inquiry Lab (BASIL). Students participating in this CURE use a combination of computational and biochemical wet-lab techniques to elucidate the function of proteins of known structure but unknown function. The goal of the project was to evaluate the efficacy of the BASIL CURE curriculum for developing students’ research abilities across implementations. Towards achieving this goal, we addressed the following four research questions (RQs): <b>RQ1</b>) How can ALOs be rigorously identified for the BASIL CURE; <b>RQ2</b>) How can the identified ALOs be used to develop a matrix that characterizes the BASIL CURE; <b>RQ3</b>) What are students’ perceptions of their knowledge, confidence and competence regarding their abilities to perform the top-rated ALOs for this CURE; <b>RQ4</b>) What are appropriate assessments for student achievement of the identified ALOs and what is the nature of student learning, and related difficulties, developed by students during the BASIL CURE? To address these RQs, this project focused on the development and use of qualitative and quantitative methods guided by constructivism and situated cognition theoretical frameworks. Data was collected using a range of instruments including, content analysis, Qualtrics surveys, open-ended questions and interviews, in order to identify ALOs and to determine student learning for the BASIL CURE. Analysis of the qualitative data was through inductive coding guided by the concept-reasoning-mode (CRM) model and the assessment triangle, while analysis of quantitative data was done by using standard statistical techniques (e.g. conducting a parried t-test and effect size). The results led to the development of a novel method for identifying ALOs, namely a process for identifying course-based undergraduate research abilities (PICURA; RQ1; Irby, Pelaez, & Anderson 2018b). Application of PICURA to the BASIL CURE resulted in the identification and rating by instructors of a wide range of ALOs, termed course-based undergraduate research abilities (CURAs), which were formulated into a matrix (RQs 2; Irby, Pelaez, & Anderson, 2018a,). The matrix was, in turn, used to characterize the BASIL CURE and to inform the design of student assessments aimed at evaluating student development of the identified CURAs (RQs 4; Irby, Pelaez, & Anderson, 2018a). Preliminary findings from implementation of the open-ended assessments in a small case study of students, revealed a range of student competencies for selected top-rated CURAs as well as evidence for student difficulties (RQ4). In this way we were able to confirm that students are developing some of the ALOs as actual learning outcomes which we term VLOs or verified learning outcomes. In addition, a participant perception indicator (PPI) survey was used to gauge students’ perceptions of their gains in knowledge, experience, and confidence during the BASIL CURE and, therefore, to inform which CURAs should be specifically targeted for assessment in specific BASIL implementations (RQ3;). These results indicate that, across implementations of the CURE, students perceived significant gains with large effect sizes in their knowledge, experience, and confidence for items on the PPI survey (RQ3;). In our view, the results of this dissertation will make important contributions to the CURE literature, as well as to the biochemistry education and assessment literature in general. More specifically, it will significantly improve understanding of the nature of student learning from CUREs and how to identify ALOs and design assessments that reveal what students actually learn from such CUREs - an area where there has been a dearth of available knowledge in the past. The outcomes of this dissertation could also help instructors and administrators identify and align assessments with the actual features of a CURE (or courses in general), use the identified CURAs to ensure the material fits departmental or university needs, and evaluate the benefits of students participating in these innovative curricula. Future research will focus on expanding the development and validation of assessments so that practitioners can better evaluate the efficacy of their CUREs for developing the research competencies of their undergraduate students and continue to render improvements to their curricula.</p> Biochemistry Education Course-based Undergraduate Research Course-based Research Experiences CURE anticipated learning outcomes ALOs ALO CURAs research abilities PICURA ALO matrix BASIL weighted-relevance WR verified learning outcomes VLOs CUREs biochemistry computational biochemistry bioinformatics biochemistry education chemistry education chemistry teaching laboratory upper-division ACE-Bio experimental competencies Authentic science inquiry chemical education assessment assessment design Student understanding student difficulties biochemistry laboratory experiments biochemistry teaching laboratory chemistry teaching laboratory chemistry teaching laboratories predicting protein function protein inquiry-based learning inquiry-based teaching content analysis open-ended survey interviews semi-structured interviews PPI participant perception indicator assessment triangle CRM conceptual reasoning mode conceptual-reasoning-mode model curriculum evaluation experimental abilities discovery skills learning objectives learning outcomes LO LOs collaborations collaborative course implementation implementation student difficuluties experimental learning experimental literacy data-representation ACE-Bio Network Likert-survey

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Étudier les fonctions des protéines avec des nanoantennes fluorescentes

Evaluation of a Novel Biochemistry Course-Based Undergraduate Research Experience (CURE)