Illuminating biomolécules : shedding light on the utility of labeling using transglutaminases

Le développement des technologies de recombinaison en biologie moléculaire fut un point tournant pour les sciences biologiques. Depuis cette découverte, diverses avancées extraordinaires qui ont un impact direct sur les humains ont pu être accomplies dans les domaines de recherches qui découlent de cette technologie. L’étude des enzymes produites en utilisant cette technique est le fondement de leurs applications éventuellement accessibles. À cet effet, la biocatalyse est un sous-domaine de l’enzymologie en développement continuel. Les chimistes et ingénieurs utilisent les composantes de systèmes biologiques ou même des systèmes complets afin de complémenter ou remplacer des méthodologies existantes. Cette thèse étudie la famille d’enzymes transglutaminase (TGase) comme biocatalyseur afin d’explorer et d’étendre l’ubiquité et les innovations rendues possibles grâce aux enzymes. Les TGases sont des enzymes versatiles. Leur homologue bactérien, la transglutaminase bactérienne (MTG), est couramment utilisé à l’échelle industrielle pour la transformation alimentaire. Depuis une dizaines d’années, de nombreux efforts ont été faits afin de trouver de nouvelles applications des TGases. En premier lieu, une revue des accomplissements, progrès et défis reliés au développement des TGases sera décrite. Les TGases sont intrinsèquement des catalyseurs de la formation de lien isopeptidiques entre une glutamine et une lysine. Par ce fait, elles ont été initialement testées dans cette thèse pour la synthèse de peptides. Une forme de l’enzyme TGase de mammifères fut en mesure de générer les composés dipeptidiques Gly-Xaa et D-Ala-Gly avec une faible conversion. La MTG possède plusieurs caractéristiques qui font de cette enzyme un candidat intéressant pour le développement de biotechnologies. Elle est stable, non dépendante d’un cofacteur et connait peu de compétition pour sa réaction catalytique inverse. La majeure partie de cette thèse porte exclusivement sur l’utilisation de la MTG. Nous avons développé et caractérisé une réaction chimio-enzymatique en un seul pot pour la conjugaison de peptides et protéines. La présence de glutathion en quantité suffisante permet de contourner l’incompatibilité de la MTG avec le cuivre et ouvre la porte à l’utilisation de la réaction de cycloaddition entre un alcyne et un azoture catalysée par le cuivre, afin d’effectuer le marquage fluorescent de protéines. L’utilisation d’autres méthodes de chimie « click » sans métaux fut aussi étudiée afin d’incorporer divers substrats protéiques. Le marquage de protéines avec la MTG fut investigué de manière combinatoire. Précisément, la ligation de Staudinger, la cycloaddition azoture-alcyne promue par la tension de cycle, ainsi que la ligation de tetrazine (TL) ont été testées. Différents niveaux de conversion ont été atteints, le plus prometteur étant celui obtenu avec la TL. Une étude par cristallographie a été effectuée afin d’élucider comment les substrats contenant une glutamine interagissent avec la MTG. Une méthode de purification alternative de la MTG a été développée afin d’atteindre ce but. Une discussion sur les stratégies et défis est présentée. Finalement, la conjugaison entre un système contenant la MTG comme biocatalyseur de marquage, le domaine B1 de la protéine G (GB1) comme substrat et d’un fluorophore contenant une amine comme sonde fut étudié. Comme deux des constituants de ce système sont des protéines, l’ingénierie d’enzyme peut être entreprise afin d’améliorer leurs propriétés. Une banque de 24 variantes de GB1 fut construite grâce à une approche semi-rationnelle afin d’investiguer quels facteurs sont déterminants pour la sélectivité de la MTG envers la glutamine. Chaque variante étudiée comportait une seule glutamine à une position variable afin d’évaluer l’impact des éléments de structure secondaire où se retrouve la glutamine. L’efficacité pour le marquage a pu être améliorée d’au moins un ordre de grandeur pour huit des substitutions étudiées. Comme chacune des structures secondaires fut marquée, il fut démontré que la MTG n’en préfère pas une en particulier. De plus, la réactivité de la MTG envers la variante I6Q-GB1 fut augmentée en créant des mutations dans son site actif. Ces résultats permettent de comprendre d’avantage la sélectivité de la MTG envers la glutamine, tout en démontrant le potentiel de cette enzyme à être modifiée afin d’être améliorée.The development of recombinant molecular biology technologies was a turning point for the biological sciences, which has since evolved into dozens upon dozens of different subfields and contributed to extraordinary advances for humans. At the core of many of these advances are the enzymes produced by these techniques, with efforts to understand their form and function laying the groundwork for their application. One of these continuously advancing subfields rooted in enzymology is biocatalysis, in which chemists and engineers embrace biological components and systems to complement, or even replace, existing methodologies. This thesis seeks to further contribute to the advancement and ubiquity of enzymes to be incorporated into future innovations. To this end, transglutaminase (TGase) is the biocatalyst selected for study. TGases are versatile enzymes, with the bacterial homolog, microbial transglutaminase (MTG) being readily used in industrial processes for years, particularly for food processing. An abundance of efforts seeking to apply TGases to other processes have been made within the last decade. We commence by reviewing the accomplishments, progress, and challenges to developing TGase towards new goals. TGase naturally catalyzes the formation of isopeptide bonds utilizing a glutamine and lysine substrates, and one of its first unconventional applications we investigated was for peptide synthesis. We determined the ability and specificity of one form of TGase for various amino acid-derived substrates, observing the formation of Gly-Xaa and D-Ala-Gly dipeptide products, albeit at a low conversion. MTG exhibits several characteristics that make it an appealing candidate for biotechnological development, such as its independence from a cofactor, little competition for its reverse catalytic reaction, and increased stability relative to mammalian TGases. Therefore, the remainder of this thesis pertains exclusively to MTG. We developed and extensively characterized a one-pot chemoenzymatic peptide and protein conjugation scheme. The presence of sufficient glutathione circumvents the incompatibility of the copper-catalyzed azide-alkyne cycloaddition with MTG owing to the presence of copper. We ultimately utilized this chemoenzymatic conjugation scheme for fluorescent protein labeling. We continue to expand upon combinatorial methods to undertake protein labeling by investigating to what extent metal-free click chemistries can be utilized in combination with MTG. Specifically, the Staudinger ligation, strain-promoted azide-alkyne cycloaddition, and tetrazine ligation (TL) were assayed on protein substrates to reveal varying levels of effective conjugation, with the TL being the most promising of the three. The details surrounding the manner in which MTG interacts with its glutamine-containing substrate remains unclear. To address this knowledge gap, we sought to pursue crystallography studies, which required the development a modified purification strategy. We discuss the strategies we investigated and the challenges surrounding such efforts. Finally, we present a conjugation system consisting of MTG as the labeling biocatalyst, the B1 domain of Protein G (GB1) as a substrate, and a small-molecule amine belonging to a recently developed class of fluorophores as a probe. As two components of this system are proteins, enzyme engineering can be applied to further improve their properties. A semi-rational approach was used to generate a 24-member GB1 library to probe the structural determinants of MTG’s glutamine selectivity. Each variant contained a single glutamine at varying positions covering all secondary structure elements, and assayed for reactivity. Eight substitutions resulting in an increased labeling efficiency of at least an order of magnitude were distributed throughout all secondary structure elements, indicating that MTG does not favor one preferentially. In addition, introducing point mutations within MTG’s active site also resulted in increased reactivity towards variant I6Q-GB1. Our results contribute further to understanding the nature of MTG’s glutamine selectivity, while simultaneously demonstrating the potential enzyme engineering has to improve and adjust this system

A Method to Accomplish the Optimal Control of Continuous Dynamical Systems with Impulse Controls via Discrete Optimal Control and Utilizing Optimal Control Theory to Explore the Emergence of Synchrony.

This research concerns the development of new optimal control methodologies and applications. In the first chapter we consider systems of ordinary differential equations subject to a restricted number of impulse controls. Examples of such systems include tumor growth, in which case the impulsive control is the administration of medication, and ecological invasion, in which case the impulse control is the release of predator species. Impulse control problems are typically solved via related partial differential equations known as quasi-variational inequalities. We show that these types of impulse control problems can be formulated as a discrete optimal control problems. Furthermore, this formulation is advantageous because it simplifies numerical calculations. In the second chapter we consider how optimal control can be used to investigate the emergence of synchrony in networks of coupled oscillators. In particular, we apply optimal control to a network of Kuramoto oscillators with time-varying coupling in order to relate network synchrony to network connectivity. To the best of our knowledge this is the first such use of optimal control theory

The Expression of Circadian Rhythms in the Non-Diapausing Mosquito Culex pipiens f. molestus

Mosquitos within the Culex pipiens species complex are primary vectors of West Nile virus in North America. The most common member of this complex in New Jersey, Cx. pipiens f. pipiens, is adapted to a temperate climate and females enter a diapause state as adults each winter. In contrast, its co-occurring sister taxon, Cx. pipiens f. molestus, is unable to enter the diapause state and both males and females continue feeding and breeding during winter months. Females additionally continue to lay eggs. The molestus form can do so because it is highly adapted to urban environments and is predominantly found in manmade underground locations. Prior studies have shown that the genes associated with circadian rhythms (i.e. ‘clock genes’) also influence the photoperiodic induction of diapause in Cx. pipiens. Here I investigated whether New World Cx. pipiens f. molestus maintains circadian rhythms despite its inability to enter a diapause state. The adult emergence of mosquitos reared in 12:12 light:dark, constant light, and constant dark had circular means in or around early scotophase (dark period). The adult emergence of mosquitos reared in the contrasting cycle of 12:12 dark:light had a circular mean after lights on, which was scotophase in the incubator. These results indicate that circadian rhythms in New World Cx. pipiens f. molestus are entrained by environmental cues during the larval period. Genetic analysis using BLASTn compared the expression of known clock genes in Cx. pipiens f. molestus and the closely related species Cx. quinquefasciatus. All known clock genes were expressed in Cx. pipiens f. molestus and suggest that the lack of diapause in these mosquitos is unlikely to be caused by major inactivating mutations in known clock genes

Social Inclusion for the United States

This working paper calls for a new policy framework -- social inclusion -- that assesses the extent to which low-paid workers fall behind the rest of the workforce, not only in terms of wages and income, but also with respect to health, education, housing, skills, advancement and opportunity. The authors discuss how the concept of social inclusion is used in the United Kingdom and how it might be adapted for use in the United States

Youth services: Promoting health, inclusion, and resiliency through libraries and library education

The ALISE Youth Services Special Interest Group (SIG) presents a panel that explores the ways in which youth services in libraries support health, inclusion, and youth resiliency. The session will begin with presentations of two papers (25 minutes each), followed by a Q&A. Attendees with then be able to share their own works in progress on these topics. The two presentations include research from Dr. Maria Cahill, Dr. Denice Adkins, and Dr. Bobbie Bushman focusing on library services for young children with disabilities and research from Dr. Denice Adkins, Dr. Beth Brendler, and Kerry Townsend on how school librarians can support youth mental health. The work of Dr. Cahill, Dr. Adkins, and Dr. Bushman will include a review of the existing literature examining library services for young children with disabilities and/or developmental delays. This review attempts to identify large gaps in the current knowledge base and instigate a call to action for the field. Dr. Adkins, Dr. Brendler, and Dr. Townsend will present preliminary findings from an IMLS-funded, mixed-methods research project, including an introduction to scalable strategies for rural public and school libraries who wish to support community health needs, an example of which is the creation of an elective course offered at the University of Missouri designed to help future librarians support teen mental health through young adult literature. The COVID-19 pandemic, subsequent interruption to in-person schooling, and existing economic and health inequalities among young people have all served to contribute to an emerging crisis in young people’s mental health and disability support services. This presentation and the subsequent attendee discussion will begin to explore the ways that the LIS field can work to address this crisis

The Changing Health of Canadian Grandparents

Fertility postponement and mortality decline are shifting the demography of the grandparent population in Canada. The ways in which the aging of the grandparent population affects families depends in large part on the health of grandparents. In this article, we document the aging of Canadian grandparents between 1985 and 2011. However, despite being older, grandparents are healthier, signaling that the compression of morbidity is outpacing the postponement of grandparenthood. This shift is partly due to the higher educational attainment of this population and partly due to secular improvements in health over time. The improved health of grandparents in Canada has important implications for intergenerational transfers and relationships

Hungry for Change: Reforming Food Distribution in India

Decrease hunger and malnutrition in Madhya Pradesh, India. A corrupt food distribution system prevents government subsidized food from getting to people. Portioning and protecting food in air tight bags will eliminate spoiling and theft.https://digitalcommons.wpi.edu/gps-posters/1132/thumbnail.jp

ICT in the classroom - Gaps between policy and practice?

En el aprendizaje de las lenguas extranjeras, obligatorio para estudiantes de 8 a 14 años según el Currículo Nacional Inglés, la necesidad de integrar la tecnología en los métodos de enseñanza ha sido evidente: “las escuelas deberían utilizar una gran variedad de recursos, incluyendo TIC, para acceder a la información y comunicarse en la lengua meta” (Departamento de Educación, 2007). El estudio muestra los motivos por los cuales los profesores hacen uso de la tecnología así como su impacto en la motivación del alumno. Descubre también una clara tendencia de usar TIC para presentar el idioma en lugar de para promover un aprendizaje autónomo y personalizado. Los alumnos expresan una satisfacción subjetiva ante la tecnología utilizada en el aula de lenguas modernas así como el deseo de hacer uso de dicha tecnología de una forma más independiente. La tecnología gira, según este estudio a pequeña escala, alrededor de la figura del profesor, quien se encarga de su uso, y son escasos los ejemplos de las posibilidades comunicativas de naturaleza autónoma descritas previamente. Las conclusiones derivan de los cuestionarios realizados por 60 estudiantes y (sus) 2 profesores, así como de las conversaciones con los profesores de dos escuelas públicas inglesasIn foreign language learning, required by the government's National Curriculum for children in England aged 8 to 14, the integration of technology into teaching methodologies has been explicit: "schools should use a range of resources, including ICT, (Information and Communication Technology) for accessing and communicating information in the target language" (Deparment for Education, 2007). This reseach investigates the role of ICT in language learning inside two English classrooms -a primary and a secondary - in order to explore the nature of motivation in educational technology from both the student and teacher perspective. The study elicits teachers' reasons for deploying the technology along with their emphases on the motivational advantages of ITC. It finds a marked tendency to use ICT for presentational, rather than autonomous or personalised learning. Children express subjective satisfaction with tecnology deployed in language learning and a desire to operate the technology more independently. Teh techonology is, from this small scale study, tecaher-led and teacher-driven, with few instances of the more autonomous communication possibilities outlined above (Department form Education, 2007). Field research findings derive from questionnaires of 60 pupils and (their) 2 teachers, as well as semi-structured interviews with teachers in two mainstream English school setting

TIC en el aula inglesa –¿huecos entre la política y la práctica?

En el aprendizaje de las lenguas extranjeras, obligatorio para estudiantes de 8 a 14 años según el Currículo Nacional Inglés, la necesidad de integrar la tecnología en los métodos de enseñanza ha sido evidente: “las escuelas deberían utilizar una gran variedad de recursos, incluyendo TIC, para acceder a la información y comunicarse en la lengua meta” (Departamento de Educación, 2007). El estudio muestra los motivos por los cuales los profesores hacen uso de la tecnología así como su impacto en la motivacióndel alumno. Descubre también una clara tendencia de usar TIC para presentar el idioma en lugar de para promover un aprendizaje autónomo y personalizado. Los alumnos expresan una satisfacción subjetiva ante la tecnología utilizada en el aula de lenguas modernas así como el deseo de hacer uso de dicha tecnología de una forma más independiente. La tecnología gira, según este estudio a pequeña escala, alrededor de la figura del profesor, quien se encarga de su uso, y son escasos los ejemplos de las posibilidades comunicativas de naturaleza autónoma descritas previamente. Las conclusiones derivan de los cuestionarios realizados por 60 estudiantes y (sus) 2 profesores, así como de las conversaciones con los profesores de dos escuelas públicas inglesas.In foreign language learning, required by the government's National Curriculum for children in England aged 8 to 14, the integration of technology into teaching methodologies has been explicit: “schools should use a range of resources, including ICT, (Information and Communication Technology) for accessing and communicating information in the target language" (Department for Education, 2007). This research investigates the role of ICT in language learning inside two English classrooms -a primary and a secondary –in order to explore the nature of motivation in educational technology from both the student and teacher perspective. The study elicits teachers' reasons for deploying the technology along with their emphases on the motivational advantages of ICT. It finds a marked tendency to use ICT for presentational, rather than autonomous or personalised learning. Children express subjective satisfaction with technology deployed in language learning and a desire to operate the technology more independently. The technology is, from this small scale study, teacher-led and teacher-driven, with few instances of the more autonomous communication possibilities outlined above (Department for Education, 2007). Field research findings derive from questionnaires of 60 pupils and (their) 2 teachers, as well as semi-structured interviews with teachers in two mainstream English school settings