Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 16/19 Archiv

Summary: Structure of the FACT chaperone domain in complex with histones H2A-H2B, and a model for FACT-mediated nucleosome reorganization Nucleosomes are the smalles unit of chromatin: two coils of DNA are wrapped around a histone octamer core, which neutralizes its charge and `packs' the lengthy molecule. Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of nucleosome remodeling machines and histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. The eukaryotic FACT complex (composed of the subunits Spt16 and Pob3) is an essential and highly conserved chaperone. It assists the progression of DNA and RNA polymerases, for example by facilitating transcriptional initiation and elongation. Further, it promotes the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription. Genetic and biochemical assays have shown that FACT's chaperone activity is crucially mediated by a direct interaction with histones H2A-H2B. However, the structural basis for how H2A-H2B are recognized and how this integrates with FACT’s other functions, including the recognition of histones H3-H4 and of other nuclear factors, is unknown. In my PhD research project, I was able to reveal the structure of the yeast chaperone domain in complex with the H2A-H2B heterodimer and show that the Spt16M module in FACT’s Spt16 subunit establishes the evolutionarily conserved H2A-H2B binding and chaperoning function. The structure shows how an alpha-helical `U-turn' motif in Spt16M interacts with the alpha-1-helix of H2B. The U-turn motif scaffolds onto a tandem pleckstrin-homology-like (PHL) module, which is structurally and functionally related to the H3-H4 chaperone Rtt106 and the Pob3M domain of FACT. Biochemical and in vivo assays validate the crystal structure and dissect the contribution of histone tails and H3-H4 toward FACT binding. My results show that Spt16M makes multiple interactions with histones, which I suggest allow the module to gradually invade the nucleosome and ultimately block the strongest interaction surface of H2B with nucleosomal DNA by binding the H2B alpha-1-helix. Together, these multiple contact points establish an extended surface that could reorganize the first 30 base-pairs of nucleosomal histone–DNA contacts. Further, I report a brief biochemical analysis of FACT’s heterodimerization domain. Its PHL fold indicates shared evolutionary origin with the H3-H4-binding Spt16M, Pob3M and Rtt106 tandem PHL modules. However, the Spt16D–Pob3N heterodimer does not bind histones, rather it connects FACT to replicative DNA polymerases. The snapshots of FACT’s engagement with H2A-H2B and structure-function analysis of all its domains lay the foundation for the systematic analysis of FACT’s vital chaperoning functions and how the complex promotes the activity of enzymes that require nucleosome reorganization.

The elimination of virus-infected cells to block viral spread is substantially conveyed by effector CD8+ T cells. Trafficking of naïve T cells into the lymph node is a crucial step for their activation to effector T cells. The entry into the lymph nodes is mediated by several cell adhesion molecules that are highly specific for given lymph nodes. For the ingress into the gut-associated lymphoid tissue (GALT) such as mesenteric lymph nodes and Peyer’s patches, the integrin α4β7 is the major homing receptor that interacts with its main ligand MAdCAM-1. On naïve T cells, α4β7 is expressed at low level, however upon activation in the GALT its expression is significantly upregulated on effector T cells. Consequently these effector T cells migrate into the gastrointestinal tract, where MAdCAM-1 is also expressed. The migration patterns of virus-specific CD8+ T cells upon viral infection have been studied in detail. In contrast, the migration of the major population of virus-unspecific CD8+ T cells, i.e. bystander-activated T cells is poorly understood. In our study, we aimed to determine how trafficking of these non-cognate, bystander-activated T cells is affected upon viral infection. We discovered that α4β7 expression was negatively regulated on bystander-activated CD8+ T cells upon injection of poly (I:C), which imitates innate immune activation upon viral infection. This effect was also observed for viral infections such as Sendai, EMCV and the mutant form of VSV virus. Furthermore, we scrutinized the direct role of IFN-α on T cells to exert α4β7 modulation. In the case of EMCV infection, IL-6 played a dominant role in the alteration of α4β7 expression. Finally, using an adoptive transfer model we could prove that the downregulation severely impacted the trafficking of T cells into the Peyer’s patches and to a lesser extent into the mesenteric lymph nodes. These findings demonstrate that a mechanism to regulate trafficking of bystander-activated T cells during viral infection exists and that this is controlled by the induction of cytokines such as IFN-α and IL-6. We hypothesize that the downregulation of α4β7 on naïve T cells functions 1) to allow space for virus-specific effector T cells to expand in the GALT and 2) to exclude bystander-activated T cells from the GALT in order to prevent mistrafficking, which could cause autoimmune diseases.

Beim kaposiformen Hämangioendotheliom handelt es sich um einen seltenen vaskulären Tumor des Säuglings- und Kindesalters. Bisher existiert keine einheitliche Behandlungsstrategie. Durch Auswertung der bisher in der Literatur veröffentlichten Fälle sowie eigener Patientendaten werden die bisher eingesetzten Therapieformen verglichen und bewertet.

Einleitung: Der Vorhofseptumdefekt (ASD) macht 6 – 10% aller ange-borenen Herzfehler (AHF) aus, im Erwachsenenalter bis zu 40%. 80% aller ASDs sind von Secundum-Typ (II). Sicherheit und Wirksamkeit des Amplatzer-Devices ASO wurden bereits bewiesen; nichtsdestotrotz traten zum Teil schwere Komplikationen auf, und der Preis ist für Entwicklungsländer zu hoch. Ein neues Device, der Nit-Occlud ASD-R, gefertigt aus einem Nitinoldraht ohne Lötstellen, mit Polyesthermembranen und Reverse-Konfiguration, entwickelt und hergestellt in Bolivien, soll auch diese Nachteile noch überwinden. Zielsetzung: Vorstellung der kurz- und mittelfristigen Ergebnisse des perkutanen Verschlusses des ASD II mit dem ASD-R in Bolivien. Methodik: Retrospektive Studie an 55 Patienten, interveniert im größten kardiologischen Zentrum in Bolivien, mit Analyse der Variablen bzgl. Sicherheit und Wirksamkeit des Devices. Ergebnisse: Alter 20,9 ± 16,6 Jahre (3 bis 67); w:m = 40:15. Defektmaß 17 ± 5,7 mm (5,4 bis 28), Device-Stent 18,5 ± 5,5 mm (6 bis 30). Geringere unerwünschte Ereignisse im Zusammenhang mit dem Device traten bei 5 Patienten auf (4 Arrhythmien), nur ein schweres: Embolisierung des trotz Protrusion freigesetzten Devices nach 12h mit chirurgischer Bergung und ASD-Verschluss. Nur in 2 Patienten traten Probleme bei der Liberation auf. Die Verschlussrate am Folgetag war 85,2%, nach 6 Monaten 100%. Schlussfolgerungen: 1. Für die Patienten dieser Studie ist der perkutane ASD-Verschluss mit Nit-Occlud ASD-R eine wirksame und sichere Methode. 2. Es ereigneten sich weder Erosion noch Perforierung, das Device zeigte sich als atraumatisch. 3. Extrem große Defekte oder ungeeignete Defektränder können andere Methoden (Chirurgie) erfordern.