Membrantopologie und funktionale Charakterisierung der Transmembrandomänen des Transportkomplexes TAP

Der humane ABC-Transporter TAP (transporter associated with antigen processing) spielt in der Antigenprozessierung und in der MHC Klasse I-vermittelten Antigenpräsentation eine zentrale Rolle. Unter ATP-Hydrolyse katalysiert der heterodimere TAP-Komplex den Transport von proteosomal degradierten Peptiden aus dem Cytosol in das ER-Lumen. Diese werden im Peptidbeladungskomplex (PLC) auf MHC Klasse I-Moleküle geladen und zur Zelloberfläche transportiert, wo sie zytotoxischen T-Zellen präsentiert werden. Die Assemblierung eines funktionalen Komplexes hängt dabei von der korrekten intra- und intermolekularen Anordnung seiner Transmembransegmente (TMS) ab. Die strukturelle Organisation des TAP-Komplexes ist schon seit vielen Jahren Gegenstand intensiver Forschung und wird in der Literatur kontrovers diskutiert. Cystein-freie Proteinvarianten dienen bei der Untersuchung der Topologie als sehr gute Hilfsmittel und wurden bereits erfolgreich zur Aufklärung der Membrantopologie von komplexen Membranproteinen, wie P-glycoprotein oder der Lactose-Permease LacY, eingesetzt (Kaback et al., 2001; Loo und Clarke, 1995). Im ersten Teil der vorliegenden Arbeit wurden humane TAP1- und TAP2-defiziente Fibroblasten, die ansonsten alle anderen Komponenten der Antigenpräsentationsmaschinerie besitzen, stabil mit der jeweiligen humanen Cystein-freien TAP1- oder TAP2-Untereinheiten transfiziert. Alle 19 natürlichen Cysteine von TAP1 und TAP2 konnten vorher durch de novo Gensynthese ausgetauscht werden. Nach erfolgreicher Expression konnte ein ATP-abhängiger Peptidtransport, der spezifisch durch den viralen Inhibitor ICP47 inhibiert wurde, nachgewiesen werden. Außerdem konnte die MHC Klasse I-Oberflächenexpression der Zellen, ein Indikator einer funktionsfähigen Antigenpräsentierung, nach Transfektion mit den Cystein-freien TAP-Untereinheiten wiederhergestellt werden. Im zweiten Abschnitt wurde die Membrantopologie des humane TAP-Transporters in einem funktionalen Peptidbeladungskomplex durch ortsspezifische Mutagenese in Kombination mit der Markierung mittels thiolspezifischer Fluorophore untersucht. Durch Co-Immunpräzipitationen und Transportstudien mit radioaktiv-markierten Peptiden konnte die Funktionalität des, in Sf9 Zellen exprimierten, Cystein-freien TAP-Komplexes gezeigt werden. Einzelne Cysteine wurden aufgrund von Hydrophobizitätsanalysen in Kombination mit Sequenzvergleichen von anderen ABC-Transportern in vorhergesagte cytosolische oder ER-luminale Schleifen der TAP1-Untereinheit eingeführt und deren Zugänglichkeit mit dem thiolspezifischen, membran-impermeablen Fluorophor Fluoreszein-5-Maleimid in semi-permeabilisierten Zellen untersucht. Es konnte zum ersten Mal experimentell gezeigt werden, dass die Transmembrandomäne (TMD) der TAP1-Untereinheit in einem funktionalen Komplex aus zehn Transmembranhelices aufgebaut ist. Die TMD lässt sich in eine für die Heterodimerisierung, die Peptidbindung und den Transport essentielle und für viele ABC-Transporter charakteristische Core-Domäne, und zusätzliche N-terminale Domänen, die für die Bindung von Tapasin und die Assemblierung des PLCs verantwortlich sind, unterteilen. Somit konnte experimentell die Membrantopologie des ABC-Transporters TAP in einem funktionalen Peptidbeladungskomplex aufgeklärt werden. Der dritte Teil der Arbeit befasste sich mit der Untersuchung der veränderten Peptidbeladungskapazität, die in Kombinationen aus wildtyp TAP1 und Cystein-freiem TAP2 beobachtet wurde. Durch ortsspezifische Mutagenese wurden einzelne Cysteine in TAP2 wiedereingeführt und deren Funktionalität in Transportstudien analysiert. Es konnte ein einzelnes Cystein in TAP2 identifiziert werden, welches die Peptidbeladungskapazität beeinflusst und durch Modifikation mit thiolspezifischen Reagenzien zum Schalter für die Peptidbindung wird. Durch Quervernetzungsstudien mit radioaktiv-markierten Peptiden konnte weiterhin ein direkter Kontakt zwischen dem Cystein und dem gebundenen und somit transportierten Peptid des TAP-Transporters nachgewiesen werden.The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the ER-lumen for subsequent loading onto MHC class I molecules. These peptide/MHC complexes are recognized at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). As a central tool to investigate the structure and function of the TAP complex, cysteine-less human TAP subunits were created by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2 by other amino acids. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to ATP-dependent peptide transport and sensitivity to the TAP-specific inhibitor ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface. In the second part of the thesis the membrane topology of human TAP1 within an assembled and functional transport complex was examined by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or ER-lumen was probed by a minimal invasive approach using membrane-impermeable, thiol-specific fluorophors in semi-permeabilized “living” cells. TAP1 contains ten transmembrane segments placing the N- and C-terminus in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ABC-transporters, and a unique N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map on the structural organization of the functional TAP complex. In the last part, the peptide binding capacity of constructs with wild-typ TAP1 and Cysteine-less TAP2 was investigated. Based on Cysteine-less TAP1 and TAP2 a set of single-cysteine constructs of TAP2 were generated and one Cysteine was identified to be involved in the alteration of the peptide binding ability. The Cysteine at position 213 could restore the peptide binding capacity. Different thiol-specific reagents to modify the single Cysteine of the construct S213C could inhibit peptide binding. Furthermore crosslinking experiments revealed a direct interaction of the peptide and the Cysteine 213

Bloomington\u27s School of Law Building Inadequate, Says Dean Harvey

The following is an interview between William B. Harvey, dean of the Indiana University School of Law and John F. Schrodt, /r., editor of the I-Witness

Five Renaissance Chronicles in Leopold von Ranke\u27s Library

This article describes the chronicles of the Middle Ages and the Renaissance that are maintained at the von Ranke Library within the Syracuse University Special Collections. The chronicles are diverse in nature, in both languages used and content respresented, covering chronologies, myths, and historical events. Ironically, the chronicles lack the objectivity that von Ranke was so fervent about, but the author argues these chronicles should not be measured against later standards of critical history

Counting oriented trees in digraphs with large minimum semidegree

Let $T$ be an oriented tree on $n$ vertices with maximum degree at most $e^{o(\sqrt{\log n})}$. If $G$ is a digraph on $n$ vertices with minimum semidegree $\delta^0(G)\geq(\frac12+o(1))n$, then $G$ contains $T$ as a spanning tree, as recently shown by Kathapurkar and Montgomery (in fact, they only require maximum degree $o(n/\log n)$). This generalizes the corresponding result by Koml\'os, S\'ark\"ozy and Szemer\'edi for graphs. We investigate the natural question how many copies of $T$ the digraph $G$ contains. Our main result states that every such $G$ contains at least $|Aut(T)|^{-1}(\frac12-o(1))^nn!$ copies of $T$, which is optimal. This implies the analogous result in the undirected case.Comment: 24 page

Gap Filling in the Plant Kingdom---Trait Prediction Using Hierarchical Probabilistic Matrix Factorization

Plant traits are a key to understanding and predicting the adaptation of ecosystems to environmental changes, which motivates the TRY project aiming at constructing a global database for plant traits and becoming a standard resource for the ecological community. Despite its unprecedented coverage, a large percentage of missing data substantially constrains joint trait analysis. Meanwhile, the trait data is characterized by the hierarchical phylogenetic structure of the plant kingdom. While factorization based matrix completion techniques have been widely used to address the missing data problem, traditional matrix factorization methods are unable to leverage the phylogenetic structure. We propose hierarchical probabilistic matrix factorization (HPMF), which effectively uses hierarchical phylogenetic information for trait prediction. We demonstrate HPMF's high accuracy, effectiveness of incorporating hierarchical structure and ability to capture trait correlation through experiments.Comment: Appears in Proceedings of the 29th International Conference on Machine Learning (ICML 2012

Students’ Attributions of Instructor Credibility as a Function of Instructors’ Out-of-Class Support

This investigation examined the impact that instructor out-of-class support (OCS) and sex differences have on students’ perceptions of instructor credibility. Participants (N = 634) were randomly assigned to one of six experimental conditions manipulating the degree to which an instructor responds with a highly supportive, moderately supportive, or non-supportive message following a hypothetically stressful situation. Multivariate analyses revealed a two-way interaction effect of instructor OCS by student sex on perceptions of instructor credibility. Significant and meaningful main effects for instructor OCS on all three dimensions of credibility (i.e., competence, trustworthiness, and caring) were also obtained, with students attributing more credibility to highly supportive instructors than to instructors who provide only moderate or no messages of support

Coparental communication, relational satisfaction, and mental health in stepfamilies

This study tested a series of actor–partner interdependence models of coparental communication, relational satisfaction, and mental health in stepfamilies. Participants included 127 couples (N = 254). Results revealed 2 actor-oriented models whereby parents’ and stepparents’ coparental communication quality positively predicted their own (but not their partners’) satisfaction and mental health. A final model revealed that parents’ relational satisfaction mediated the effect of coparental communication on their own mental health. A similar pattern emerged for stepparents, although coparental communication continued to have a direct, positive effect on stepparents’ mental health. Importantly, parents’ coparental communication produced an inverse partner effect on stepparents’ mental health, highlighting the potential stress stepparents may experience as they are called upon to help raise their spouse’s offspring

Failing to Learn, Learning to Fail: Strategies to Create Empowered and Independent Learners

It’s hard to learn if we never make mistakes. The error. The stumble. The near win. These are all powerful self-teachers. Experts, in fact, guide themselves down the error-filled road to mastery. Pierce Brown (2016), in the epilogue to his novel Morning Star, provides a profound axiom for his readers: “Everything grand is made from a series of ugly little moments . . . All the works of people you and I admire sit atop a foundation of failures” (p. 523). This is the mindset we lose too often in the K-12 classroom. However, the culture of speed and the need to cover content persists, even in the chaotic aftermath of the recent school years where students juggled theuncertainties of in-person and remote learning. Many students navigated these experiences feeling like failures. Yet voices from inside and outside the education realm celebrate the same truth: failure is a key element of meaningful learning. As teachers, it is our responsibility to create learning environments that illustrate this truth in action. By using authentic examples from real-world innovators and creators, we send signals to our students that risks are the norm, uncertainty is an opportunity, and learning is not linear. When teachers surround students with proof that failure is a powerful learning tool, students will be more likely to start the unlearningprocess and embrace failing to learn, learning to fail as a way to navigate themselves down new paths toward deeper learning